Rhodococcusfascians is a nocardiform bacteria that induces leafy galls (fasciation) on dicotyledonous and several monocotyledonous plants. The wild-type strain D188 contained a conjugative, 200 kb linear extrachromosomal element, pFiD188. Linear plasmid-cured strains were avirulent and reintroduction of this linear element restored virulence. Pulsed field electrophoresis indicated that the chromosome might also be a linear molecule of 4 megabases. Three loci involved in phytopathogenicity have been identified by insertion mutagenesis of this Fi plasmid. Inactivation of the fas locus resulted in avirulent strains, whereas insertions in the two other loci affected the degree of virulence, yielding attenuated (att) and hypervirulent (hyp) bacteria. One of the genes within the fas locus encoded an isopentenyltranferase (IPT) with low homology to analogous proteins from Gram-negative phytopathogenic bacteria. IPT activity was detected after expression of this protein in Escherichia coli cells. In R.fascians, ipt expression could only be detected in bacteria induced with extracts from fasciated tissue. R.fascians strains without the linear plasmid but containing this fas locus alone could not provoke any phenotype on plants, indicating additional genes from the linear plasmid were also essential for virulence. These studies, the first genetic analysis of the interaction of a Gram-positive bacterium with plants, suggest that a novel mechanism for plant tumour induction has evolved in R.fascians independently from the other branches of the eubacteria. Images PMID:1547783

Rhodococcusfascians, a phytopathogen that alters plant development, inflicts significant losses in plant production around the world. We report here the complete genome sequence of R. fascians D188, a well-characterized model isolate, and Rhodococcus species PBTS (pistachio bushy top syndrome) 1 and 2, which were shown to be responsible for a disease outbreak in pistachios. PMID:27284129

Full Text Available The accurate diagnosis of diseases caused by pathogenic bacteria requires a stable species classification. Rhodococcusfascians is the only documented member of its ill-defined genus that is capable of causing disease on a wide range of agriculturally important plants. Comparisons of genome sequences generated from isolates of Rhodococcus associated with diseased plants revealed a level of genetic diversity consistent with them representing multiple species. To test this, we generated a tree based on more than 1700 homologous sequences from plant-associated isolates of Rhodococcus, and obtained support from additional approaches that measure and cluster based on genome similarities. Results were consistent in supporting the definition of new Rhodococcus species within clades containing phytopathogenic members. We also used the genome sequences, along with other rhodococcal genome sequences to construct a molecular phylogenetic tree as a framework for resolving the Rhodococcus genus. Results indicated that Rhodococcus has the potential for having 20 species and also confirmed a need to revisit the taxonomic groupings within Rhodococcus.

Three virulence loci (fas, att, and hyp) of Rhodococcusfascians D188 have been identified on a 200-kb conjugative linear plasmid (pFiD188). The fas locus was delimited to a 6.5-kb DNA fragment by insertion mutagenesis, single homologous disruptive recombination, and in trans complementation of different avirulent insertion mutants. The locus is arranged as a large operon containing six open reading frames whose expression is specifically induced during the interaction with host plants. One predicted protein is homologous to P-450 cytochromes from actinomycetes. The putative ferredoxin component is of a novel type containing additional domains homologous to transketolases from chemoautotrophic, photosynthetic, and methylotrophic microorganisms. Genetic analysis revealed that fas encodes, in addition to the previously identified ipt, at least two new genes that are involved in fasciation development, one of which is only required on older tobacco plants. PMID:8169198

Full Text Available AIMS: Though plant metabolic changes are known to occur during interactions with bacteria, these were rarely challenged for pharmacologically active compounds suitable for further drug development. Here, the occurrence of specific chemicals with antiproliferative activity against human cancer cell lines was evidenced in hyperplasia (leafy galls induced when plants interact with particular phytopathogens, such as the Actinomycete Rhodococcusfascians. METHODS: We examined leafy galls fraction F3.1.1 on cell proliferation, cell division and cytoskeletal disorganization of human cancer cell lines using time-lapse videomicroscopy imaging, combined with flow cytometry and immunofluorescence analysis. We determined the F3.1.1-fraction composition by gas chromatography coupled to mass spectrometry. RESULTS: The leafy galls induced on tobacco by R. fascians yielded fraction F3.1.1 which inhibited proliferation of glioblastoma U373 cells with an IC50 of 4.5 µg/mL, F.3.1.1 was shown to increase cell division duration, cause nuclear morphological deformations and cell enlargement, and, at higher concentrations, karyokinesis defects leading to polyploidization and apoptosis. F3.1.1 consisted of a mixture of isomers belonging to the cembrenoids. The cellular defects induced by F3.1.1 were caused by a peculiar cytoskeletal disorganization, with the occurrence of fragmented tubulin and strongly organized microtubule aggregates within the same cell. Colchicine, paclitaxel, and cembrene also affected U373 cell proliferation and karyokinesis, but the induced microtubule rearrangement was very different from that provoked by F3.1.1. Altogether our data indicate that the cembrenoid isomers in F3.1.1 have a unique mode of action and are able to simultaneously modulate microtubule polymerization and stability.

Full Text Available The growth-promoting bacteria in plants (BGPB are a group of different species of bacteria can increase plant growth and productivity. Which can benefit plants through their own bacterial metabolism (phosphate solubilizing, producing hormones or fixing nitrogen. At present, desertification is a growing phenomenon worldwide, afforestation is one of the common solutions to combat this problem. Trees for reforestation are initially grown in greenhouses or nurseries. Among numerous reforestation practices, there is an alternative that inoculation with PGPB. Is a forest species that is endemic Olneya tesota Sonoran Desert, which is in danger of extinction. The objective was to evaluate the effect of bacteria growth promoter in plants with Rhodococcuis fascians and Azospirillum halopraeferens on germination and emergence of Ironwood under four salt concentrations (0, 0.25, 0.5 and 0.75 M NaCl under greenhouse conditions. Were obtained ironwood seeds in the region of Santa Ana, Sonora. Under greenhouse conditions was evaluated emergence percentage, germination rate, height, plant root length, fresh and dry weight of plant, number of bacterial cells attached to the root system, fresh and dry weight of the root. The results indicate that the germination percentage and other variables evaluated decreased as salinity increases. However, these changed positively to inoculation with bacteria R. fascians and A. halopraeferens.

Four series of azidopurines have been synthesized and tested for cytokinin activity in the tobacco callus bioassay: 2- and 8-azido-N6-benzyladenines, -N6-(Δ2-isopentenyl)adenines, and -zeatins, and N6-(2- and 4-azidobenzyl)adenines. The compounds having 2-azido substitution on the adenine ring are as active as the corresponding parent compounds, while those with 8-azido substitution are about 10 or more times as active. The 8-azidozeatin, which is the most active cytokinin observed, exhibited higher than minimal detectable activity at 1.2 × 10−5 micromolar, the lowest concentration tested. The shape of the growth curve indicates that even a concentration as low as 5 × 10−6 micromolar would probably be effective. By comparison, the lowest active concentration ever reported for zeatin has been 5 × 10−5 micromolar, representing a sensitivity rarely attained. All of the azido compounds have been submitted to photolysis in aqueous ethanol, and the photoproducts have been detected and identified by low and high resolution mass spectrometry. They are rationalized as products of abstraction and insertion reactions of the intermediate nitrenes. The potential of the major released products as cytokinins was also assessed by bioassay. 2-Azido-N6-(Δ2-isopentenyl)adenine competed with [14C]kinetin for the cytokinin-binding protein isolated from wheat germ. When the azido compound was photolysed in the presence of this protein, its attachment effectively blocked the binding of [14C]kinetin. PMID:16661017

Cytokinins are adenine derivatives with an isoprenoid side chain and play an essential role in plant development. Plant isopentenyltransferases that catalyze the first and rate-limiting steps of cytokinin biosynthesis have recently been identified. Unlike bacterial enzymes, which catalyze the transfer of the isopentenyl moiety from dimethylallyldiphosphate (DMAPP) to the N(6) position of adenosine 5'-monophosphate (AMP), plant enzymes catalyze the transfer of the isopentenyl moiety from DMAPP preferentially to ATP and to ADP. The isopentenylated side chain is hydroxylated to form zeatin-type cytokinins. An alternative pathway, in which a hydroxylated side chain is directly added to the N(6) position of the adenine moiety, has also been suggested.

wheat enzyme is a monomer 60 kDa, its N-terminal amino-acid sequence shows similarity to hypothetical cytokinin oxidase genes from Arabidopsis thaliana, but not to the enzyme from maize. N-6-isopentenyl-2-(2-hydroxyethylamino)-9-methyladenine is the best substrate from all the cytokinins tested...

Rhodococcus defluvii strain Ca11T was isolated from a bioreactor involved in extensive phosphorus removal. We have sequenced the whole genome of this strain and our comparative genomic and phylogenetic analyses confirm its close relatedness with Rhodococcus equi (Rhodococcus hoagii) strains, which share >80% of the gene content. The R. equi virulence plasmid is absent though most of the chromosomal R. equi virulence-associated genes are present in R. defluvii Ca11T. These data suggest that al...

Rhodococcus defluvii strain Ca11(T) was isolated from a bioreactor involved in extensive phosphorus removal. We have sequenced the whole genome of this strain, and our comparative genomic and phylogenetic analyses confirm its close relatedness with Rhodococcus equi (Rhodococcus hoagii) strains, which share >80 % of the gene content. The R. equi virulence plasmid is absent though most of the chromosomal R. equi virulence-associated genes are present in R. defluvii Ca11(T). These data suggest that although R. defluvii is an environmental organism, it has the potential to colonize animal hosts.

Full Text Available Rhodococcus equi is an uncommon human pathogen known to cause lung infections in immunocompromised patients. We report two cases of Rhodococcus infections in immunocompetent individuals, who were treated successfully.

The roots and stems of dicotyledonous plants thicken by the cell proliferation in the cambium. Cambial proliferation changes in response to environmental factors; however, the molecular mechanisms that regulate cambial activity are largely unknown. The quadruple Arabidopsis thaliana mutant atipt1;3;5;7, in which 4 genes encoding cytokinin biosynthetic isopentenyltransferases are disrupted by T-DNA insertion, was unable to form cambium and showed reduced thickening of the root and stem. The atipt3 single mutant, which has moderately decreased levels of cytokinins, exhibited decreased root thickening without any other recognizable morphological changes. Addition of exogenously supplied cytokinins to atipt1;3;5;7 reactivated the cambium in a dose-dependent manner. When an atipt1;3;5;7 shoot scion was grafted onto WT root stock, both the root and shoot grew normally and trans-zeatin-type (tZ-type) cytokinins in the shoot were restored to WT levels, but isopentenyladenine-type cytokinins in the shoot remained unchanged. Conversely, when a WT shoot was grafted onto an atipt1;3;5;7 root, both the root and shoot grew normally and isopentenyladenine-type cytokinins in the root were restored to WT levels, but tZ-type cytokinins were only partially restored. Collectively, it can be concluded that cytokinins are important regulators of cambium development and that production of cytokinins in either the root or shoot is sufficient for normal development of both the root and shoot.

Cytokinins are plant hormones implicated in diverse and essential processes in plant growth and development, and key genes for the metabolism and actions of cytokinins have recently been identified. Cytokinins are perceived by three histidine kinases--CRE1/WOL/AHK4, AHK2, and AHK3--which initiate intracellular phosphotransfer. The final destination of the transferred phosphoryl groups is response regulators. The type-B Arabidopsis response regulators (ARRs) are DNA-binding transcriptional activators that are required for cytokinin responses. On the other hand, the type-A ARRs act as repressors of cytokinin-activated transcription. How phosphorelay regulate response regulators and how response regulators control downstream events are open questions and discussed in this review.

Mycobacterium tuberculosis (M. tuberculosis) resides mainly inside macrophages, which produce nitric oxide (NO) to combat microbial infections. Earlier studies revealed that proteasome-associated genes are required for M. tuberculosis to resist NO via a previously uncharacterized mechanism. Twelve years later, we elucidated the link between proteasome function and NO resistance in M. tuberculosis in Molecular Cell, 57 (2015), pp. 984-994. In a proteasome degradation-defective mutant, Rv1205, a homologue of the plant enzyme LONELY GUY (LOG) that is involved in the synthesis of phytohormones called cytokinins, accumulates and as a consequence results in the overproduction of cytokinins. Cytokinins break down into aldehydes that kill mycobacteria in the presence of NO. Importantly, this new discovery reveals for the first time that a mammalian bacterial pathogen produces cytokinins and leaves us with the question: why is M. tuberculosis, an exclusively human pathogen, producing cytokinins?

Five cases of Rhodococcus equi infection in dogs were identified from 2003 to 2014. Three of the dogs had severe, internal lesions attributable to R. equi that have not been previously described: endophthalmitis, endocarditis, and suppurative pleuropneumonia. Isolates from 4 of the dogs were analyzed by polymerase chain reaction for Rhodococcus virulence-associated plasmid (vap) genes. One isolate was vapA-positive, 2 lacked a virulence plasmid, and 1 carried the novel vapN-associated plasmid (pVAPN) recently characterized in bovine isolates. The pVAPN plasmid has not been described in isolates cultured from companion animals. Four of the dogs either were receiving immunosuppressive drugs or had endocrinopathies. R. equi has the potential to cause significant infections in dogs, and immunocompromised animals should be considered at risk for infection.

Plant hormones regulate many aspects of plant growth and development. Both auxin and cytokinin have been known for a long time to act either synergistically or antagonistically to control several significant developmental processes, such as the formation and maintenance of meristem. Over the past few years, exciting progress has been made to reveal the molecular mechanisms underlying the auxin-cytokinin action and interaction. In this review, we shall briefly discuss the major progress made in auxin and cytokinin biosynthesis, auxin transport, and auxin and cytokinin signaling.The frameworks for the complicated interaction of these two hormones in the control of shoot apical meristem and root apical meristem formation as well as their roles in in vitro organ regeneration are the major focus of this review.

A plasmid transformation system for Rhodococcus was developed using an Escherichia coli-Rhodococcus shuttle plasmid. Rhodococcus sp. H13-A contains three cryptic indigenous plasmids, designated pMVS100, pMVS200 and pMVS300, of 75, 19.5 and 13.4 kilobases (Kb), respectively. A 3.8 Kb restriction fragment of pMVS300 was cloned into pIJ30, a 6.3 Kb pBR322 derivative, containing the E. coli origin of replication (ori) and ampicillin resistance determinant (bla) as well as a Streptomyces gene for thiostrepton resistance, tsr. The resulting 10.1 Kb recombinant plasmid, designated pMVS301, was isolated from E. coli DH1 (pMVS301) and transformed into Rhodococcus sp. AS-50, a derivative of strain H13-A, by polyethylene glycol-assisted transformation of Rhodococcus protoplasts and selection for thiostrepton-resistant transformants. This strain was deposited with the ATCC on Feb. 1, 1988 and assigned ATCC 53719. The plasmid contains the Rhodococcus origin of replication. The plasmid and derivatives thereof can therefore be used to introduce nucleic acid sequences to and from Rhodococcus for subsequent expression and translation into protein. The isolated origin of replication can also be used in the construction of new vectors. 2 figs.

Water stress applied to the plant shoot through enhanced evaporative demands reduced cytokinin activity in extracts of xylem exudate and leaves. This reduction resembled the changes in cytokinin activity caused by water stress applied to the root. Cytokinin activity in detached wilting leaves decreased rapidly. Recovery took place after several hours in a humid chamber. Experiments with 14C-kinetin indicated that the mechanism of the inactivation and its reversal involve a chemical transformation of the cytokinin molecule. PMID:16657585

Full Text Available The aim of the research was to study the cytokinins production by medicinal basidial mushrooms. Cytokinins were for the first time identified and quantified in mycelial biomass of six species (Ganoderma lucidum, Trametes versicolor, Fomitopsis officinalis, Pleurotus nebrodensis, Grifola frondosa, Sparassis crispa using HPLC. Trans- and cis-zeatin, zeatin riboside, zeatin-O-glucoside, isopentenyladenosine, isopentenyladenine were found but only one species (G. lucidum, strain 1900 contained all these substances. The greatest total cytokinin quantity was detected in F. officinalis, strain 5004. S. crispa, strain 314, and F. officinalis, strain 5004, mycelial biomass was revealed to have the highest level of cytokinin riboside forms (zeatin riboside and isopentenyladenosine. The possible connection between medicinal properties of investigated basidiomycetes and of cytokinins is discussed. S. crispa, strain 314, and F. officinalis, strain 5004, are regarded as promising species for developing biotechnological techniques to produce biologically active drugs from their mycelial biomass. As one of the potential technological approaches there is proposed fungal material drying.

Full Text Available In flowering plants, the arrangement of flowers on a stem becomes an inflorescence, and a huge variety of inflorescence architecture occurs in nature. Inflorescence architecture also affects crop yield. In simple inflorescences, flowers form on a main stem; by contrast, in compound inflorescences, flowers form on branched stems and the branching pattern defines the architecture of the inflorescence. In this review, we highlight recent findings on the regulation of inflorescence architecture by cytokinin plant hormones. Results in rice and Arabidopsis show that although these two species have distinct inflorescence architectures, cytokinins have a common effect on inflorescence branching. Based on these studies, we discuss how cytokinins regulate distinct types of inflorescence architecture through their effect on meristem activities.

In flowering plants, the arrangement of flowers on a stem becomes an inflorescence, and a huge variety of inflorescence architecture occurs in nature. Inflorescence architecture also affects crop yield. In simple inflorescences, flowers form on a main stem; by contrast, in compound inflorescences, flowers form on branched stems and the branching pattern defines the architecture of the inflorescence. In this review, we highlight recent findings on the regulation of inflorescence architecture by cytokinin plant hormones. Results in rice (Oryza sativa) and Arabidopsis thaliana show that although these two species have distinct inflorescence architectures, cytokinins have a common effect on inflorescence branching. Based on these studies, we discuss how cytokinins regulate distinct types of inflorescence architecture through their effect on meristem activities.

Rhodococcus equi is an important etiologic agent of respiratory- and non-respiratory tract infections, diseases of animals and humans. Therapy includes the use of various group of chemotherapeutic agents, however resistance acquirement is quite common. To date there is no preferred treatment protocol for infections caused by isolates resistant to macrolides and rifampicin. The resistance acquirement is a result of many molecular mechanisms, some of which include alterations in the cell envelope composition and structure, activity of the efflux pumps, enzymatic destruction or inactivation of antibiotics, and changes in the target site. This paper contains an overview of antimicrobial susceptibility of R. equi, and explains the possible molecular mechanisms responsible for antimicrobial resistance in this particular microorganism.

The transcriptomics approach to study gene expression in root hairs from M. truncatula has shed light on the developmental events during rhizobial infection and the underlying hormone responses. This approach revealed the induction of several cyclins and an aurora kinase which suggests that the cell-division machinery plays a role in rhizobial infection. Changes in the cell cycle in plants are governed by hormones, in particular auxin and cytokinin. Through gene expression and genetic analyses, we have shown auxin plays a role during rhizobial infection. Here we provide further analysis of the data showing the induction of a set of cytokinin signaling components. These include genes encoding 2 cytokinin-activating enzymes, the cytokinin receptor CRE1, and 5 type-A cytokinin response regulators. We discuss the possible interactions between auxin and cytokinin signaling during the infection process. We also consider a potential role for cytokinin signaling in rhizobial attachment. PMID:26176899

A simple and rapid colorimetric assay for cytokinin oxidase is described. The assay is based on the formation of a Schiff base between the enzymatic reaction product 3-methyl-2-butenal and p-aminophenol. The assay is effective in the submicromolar concentration range and can be used in crude plant extracts as well as in more highly purified preparations.

A Gram-stain-positive, non-endospore-forming rod-shaped bacterium, strain Ca11(T), was isolated from a bioreactor with extensive phosphorus removal and was studied in detail for its taxonomic allocation. 16S rRNA gene sequence analysis revealed closest sequence similarity of the strain to type strains of [Corynebacterium hoagii] and Rhodococcus equi (98.9%), Rhodococcus koreensis and Rhodococcus wratislaviensis (both 98.4%), Rhodococcus opacus and Rhodococcus canchipurensis (both 98.0%) followed by Rhodococcus kunmingensis and Rhodococcus imtechensis (97.7%). Phylogenetic trees showed a distinct clustering of strain Ca11(T) with the type strains of [C. hoagii], R. equi, and R. kunmingensis separate to all other species of the genus Rhodococcus. The quinone system of strain Ca11(T) was composed of dihydrogenated menaquinones with 8 (major amount) as well as 7 and 6 isoprenoid units [MK-8(H2), MK-7(H2), MK-6(H2)]. The polar lipid profile consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol mannoside, one unknown phospholipid and an unidentified glycolipid. The fatty acid profile was similar to that reported for R. equi and contained major amounts of C16:0, C18:1ω9c and 10-methyl C18:0, supporting the allocation of the strain to the genus Rhodococcus. Physiological and biochemical characterization and DNA-DNA hybridization with type strains of the most closely related species allowed clear phenotypic and genotypic differentiation of the isolate. On the basis of these results, strain Ca11(T) ( = DSM 45893(T) = LMG 27563(T)) represents a novel species of the genus Rhodococcus, with the proposed name Rhodococcus defluvii sp. nov. In addition, a polyphasic taxonomic analysis of [Corynebacterium hoagii] DSM 20295(T) and Rhodococcus equi DSM 20307(T) indicated that the two strains belong to the same species, for which the name Rhodococcus hoagii comb. nov. takes priority, according to the Rules of the

Full Text Available Cytokinins are involved in plant cell proliferation leading to plant growth and morphogenesis. The size, activity and maintenance of the shoot apical meristem (SAM are defined by a balanced rate of mitotic cell divisions and functional cell differentiation that are controlled by cytokinins. In order to investigate the effect of exogenous cytokinin on SAM, morpho-anatomical changes in the shoot apices of tobacco treated with benzyladenine (BA were compared to those of untreated control plants.

Cytokinins are a class of mitogenic plant hormones that play an important role in most aspects of plant development, including shoot and root growth, vascular and photomorphogenic development and leaf senescence. A model for cytokinin perception and signaling has emerged that is similar to bacterial two-component phosphorelays. In this model, binding of cytokinin to the extracellular domain of the Arabidopsis histidine kinase (AHKs) receptors induces autophosphorylation within the intracellular histidine-kinase domain. The phosphoryl group is subsequently transferred to cytosolic Arabidopsis histidine phosphotransfer proteins (AHPs), which have been suggested to translocate to the nucleus in response to cytokinin treatment, where they then transfer the phosphoryl group to nuclear-localized response regulators (Type-A and Type-B ARRs). We examined the effects of cytokinin on AHP subcellular localization in Arabidopsis and, contrary to expectations, the AHPs maintained a constant nuclear/cytosolic distribution following cytokinin treatment. Furthermore, mutation of the conserved phosphoacceptor histidine residue of the AHP, as well as disruption of multiple cytokinin signaling elements, did not affect the subcellular localization of the AHP proteins. Finally, we present data indicating that AHPs maintain a nuclear/cytosolic distribution by balancing active transport into and out of the nucleus. Our findings suggest that the current models indicating relocalization of AHP protein into the nucleus in response to cytokinin are incorrect. Rather, AHPs actively maintain a consistent nuclear/cytosolic distribution regardless of the status of the cytokinin response pathway.

Cytokinins are a class of mitogenic plant hormones that play an important role in most aspects of plant development, including shoot and root growth, vascular and photomorphogenic development and leaf senescence. A model for cytokinin perception and signaling has emerged that is similar to bacterial two-component phosphorelays. In this model, binding of cytokinin to the extracellular domain of the Arabidopsis histidine kinase (AHKs) receptors induces autophosphorylation within the intracellular histidine-kinase domain. The phosphoryl group is subsequently transferred to cytosolic Arabidopsis histidine phosphotransfer proteins (AHPs), which have been suggested to translocate to the nucleus in response to cytokinin treatment, where they then transfer the phosphoryl group to nuclear-localized response regulators (Type-A and Type-B ARRs). We examined the effects of cytokinin on AHP subcellular localization in Arabidopsis and, contrary to expectations, the AHPs maintained a constant nuclear/cytosolic distribution following cytokinin treatment. Furthermore, mutation of the conserved phosphoacceptor histidine residue of the AHP, as well as disruption of multiple cytokinin signaling elements, did not affect the subcellular localization of the AHP proteins. Finally, we present data indicating that AHPs maintain a nuclear/cytosolic distribution by balancing active transport into and out of the nucleus. Our findings suggest that the current models indicating relocalization of AHP protein into the nucleus in response to cytokinin are incorrect. Rather, AHPs actively maintain a consistent nuclear/cytosolic distribution regardless of the status of the cytokinin response pathway.

The puzzle piece-shaped Arabidopsis leaf pavement cells (PCs) with interdigitated lobes and indents is a good model system to investigate the mechanisms that coordinate cell polarity and shape formation within a tissue.Auxin has been shown to coordinate the interdigitation by activating ROP GTPase-dependent signaling pathways.To identify additional components or mechanisms,we screened for mutants with abnormal PC morphogenesis and found that cytokinin signaling regulates the PC interdigitation pattern.Reduction in cytokinin accumulation and defects in cytokinin signaling (such as in ARR7-over-expressing lines,the ahk3cre1 cytokinin receptor mutant,and the ahp12345 cytokinin signaling mutant) enhanced PC interdigitation,whereas over-production of cytokinin and over-activation of cytokinin signaling in an ARR20 over-expression line delayed or abolished PC interdigitation throughout the cotyledon.Genetic and biochemical analyses suggest that cytokinin signaling acts upstream of ROPs to suppress the formation of interdigitated pattern.Our results provide novel mechanistic understanding of the pathways controlling PC shape and uncover a new role for cytokinin signaling in cell morphogenesis.

The growth-promoting bacteria in plants (BGPB) are a group of different species of bacteria can increase plant growth and productivity. Which can benefit plants through their own bacterial metabolism (phosphate solubilizing, producing hormones or fixing nitrogen). At present, desertification is a growing phenomenon worldwide, afforestation is one of the common solutions to combat this problem. Trees for reforestation are initially grown in greenhouses or nurseries. Among numerous reforestatio...

The models explaining root gravitropism propose that the growth response of plants to gravity is regulated by asymmetric distribution of auxin (indole-3-acetic acid, IAA). Since cytokinin has a negative regulatory role in root growth, we suspected that it might function as an inhibitor of tropic root elongation during gravity response. Therefore, we examined the free-bioactive-cytokinin-dependent ARR5::GUS expression pattern in root tips of transformants of Arabidopsis thaliana (L.) Heynh., visualized high cytokinin concentrations in the root cap with specific monoclonal antibodies, and complemented the analyses by external application of cytokinin. Our findings show that mainly the statocytes of the cap produce cytokinin, which may contribute to the regulation of root gravitropism. The homogenous symmetric expression of the cytokinin-responsive promoter in vertical root caps rapidly changed within less than 30 min of gravistimulation into an asymmetrical activation pattern, visualized as a lateral, distinctly stained, concentrated spot on the new lower root side of the cap cells. This asymmetric cytokinin distribution obviously caused initiation of a downward curvature near the root apex during the early rapid phase of gravity response, by inhibiting elongation at the lower side and promoting growth at the upper side of the distal elongation zone closely behind the root cap. Exogenous cytokinin applied to vertical roots induced root bending towards the application site, confirming the suspected inhibitory effect of cytokinin in root gravitropism. Our results suggest that the early root graviresponse is controlled by cytokinin. We conclude that both cytokinin and auxin are key hormones that regulate root gravitropism.

Cytokinins are distributed through the vascular system and trigger responses of target cells via receptor-mediated signal transduction. Perception and transduction of the signal can occur at the plasma membrane or in the cytosol. The signal is terminated by the action of extra- or intracellular cytokinin oxidases. While radiotracer studies have been used to study transport and metabolism of cytokinins in plants, little is known about the kinetic properties of cytokinin transport. To provide a reference dataset, radiolabeled trans-zeatin (tZ) was used for uptake studies in Arabidopsis (Arabidopsis thaliana) cell culture. Uptake kinetics of tZ are multiphasic, indicating the presence of both low- and high-affinity transport systems. The protonophore carbonyl cyanide m-chlorophenylhydrazone is an effective inhibitor of cytokinin uptake, consistent with H(+)-mediated uptake. Other physiological cytokinins, such as isopentenyl adenine and benzylaminopurine, are effective competitors of tZ uptake, whereas allantoin has no inhibitory effect. Adenine competes for zeatin uptake, indicating that the degradation product of cytokinin oxidases is transported by the same systems. Comparison of adenine and tZ uptake in Arabidopsis seedlings reveals similar uptake kinetics. Kinetic properties, as well as substrate specificity determined in cell cultures, are compatible with the hypothesis that members of the plant-specific purine permease family play a role in adenine transport for scavenging extracellular adenine and may, in addition, be involved in low-affinity cytokinin uptake.

New approaches are described in this thesis towards the syntheses of phosphorylated and alanylated cytokinins.In chapter 1 a general picture of the stucture of cytokinins, their occurence in nature, their biological synthesis, their effects on plants and their chemical synthesis is described.A liter

A polyphasic study was undertaken to clarify the taxonomic position of the type strain (N805T) of Tsukamurella wratislaviensis. This organism showed a combination of phenotypic properties, notably chemotaxonomic markers, consistent with its classification in the genus Rhodococcus. Comparative 16S rDNA sequencing studies indicated that strain 805T falls into the Rhodococcus erythropolis subclade, where it forms a monophyletic group with the type strains of Rhodococcus opacus and Rhodococcus percolatus. The close relationship between these strains was underpinned by the results of mycolic acid analyses. However, strain N805T was distinguished from the R. opacus and R. percolatus strains in DNA-DNA pairing experiments and by using a range of phenotypic properties. In light of these studies, it is clear that strain N805T is misclassified in the genus Tsukamurella. It is, therefore, proposed that Tsukamurella wratislaviensis Goodfellow et al. 1995 be transferred to the genus Rhodococcus as Rhodococcus wratislaviensis comb. nov..

Rhodococcus equi is an aerobic, gram-positive, non-motile pleomorphic bacillus infecting immunocompromised patients. Forty-nine cases of Rhodococcus equi infection have been reported, mainly in patients infected with the human immunodeficiency virus (HIV). A case in which Rhodococcus equi caused...... severe pulmonary infection, the most common presentation, is described. Clinically, patients have symptoms of pneumonia with hemoptysis as a prominent feature. X-ray will often show a cavitating upper-lobe infiltrate, resembling infection with mycobacteria. Rhodococcus equi is easily cultured from blood......-term antibiotic treatment with erythromycin plus rifampicin, or vancomycin in combination with either of these antibiotics....

The treatment of 4-days-old wheat seedlings with methyl jasmonate (MeJA) in concentration optimal for their growth (0.1 μM) resulted in a rapid transient almost two-fold increase in the level of cytokinins (CKs). MeJA-induced accumulation of CKs was due to inhibition of both cytokinin oxidase (CKX) (cytokinin oxidase/dehydrogenase, EC 1.5.99.12) gene expression and activity of this enzyme. Pretreatment of wheat seedlings with MeJA decreased the growth-retarding effect of sodium chloride salinity and accelerated growth recovery after withdrawal of NaCl from the incubation medium. We speculate that this protective effect of the hormone might be due to MeJA's ability to prevent the salinity-induced decline in CK concentration that was caused by inhibition of gene expression and activity of CKX in wheat seedlings. The data might indicate an important role for endogenous cytokinins in the implementation of growth-promoting and protective effects of exogenous MeJA application on wheat plants.

Mycotoxins are secondary fungal metabolites that may have mutagenic, carcinogenic, cytotoxic and endocrine disrupting effects. These substances frequently contaminate agricultural commodities despite efforts to prevent them, so successful detoxification tools are needed. The application of microorganisms to biodegrade mycotoxins is a novel strategy that shows potential for application in food and feed processing. In this study we investigated the mycotoxin degradation ability of thirty-two Rhodococcus strains on economically important mycotoxins: aflatoxin B1, zearalenone, fumonisin B1, T2 toxin and ochratoxin A, and monitored the safety of aflatoxin B1 and zearalenone degradation processes and degradation products using previously developed toxicity profiling methods. Moreover, experiments were performed to analyse multi-mycotoxin-degrading ability of the best toxin degrader/detoxifier strains on aflatoxin B1, zearalenone and T2 toxin mixtures. This enabled the safest and the most effective Rhodococcus strains to be selected, even for multi-mycotoxin degradation. We concluded that several Rhodococcus species are effective in the degradation of aromatic mycotoxins and their application in mycotoxin biodetoxification processes is a promising field of biotechnology.

Full Text Available Today, owing to the versatile functionality and physiological importance of the phytohormone cytokinin (Ck is a major focus of attention in contemporary wide areas of plant science. Cytokinins (Cks have implicated in diverse essential processes of plant growth and development as well as in regulation of key genes responsible for the metabolism and activities of plants. Cytokinin interact in a complex manner to control a myriad of aspects related to growth, development and differentiation and its deficiency also causes pleiotropic developmental changes such as reduced shoot and increased root growth. Cytokinin signaling involves His Kinase receptors that perceive cytokinin and transmit the signal via a multi-step phosphorelay similar to bacterial two-component signaling system. Also, this review present a scheme for homeostatic regulation of endogenous cytokinins level in terms of the described mechanism of cytokinin action including its receptors and steps involved in regulation of gene expression at the post-transcriptional level and its role in whole plant as well as cell division. In addition, we also demonstrate a wide variety of biological effects including those on gene expression, inhibition of auxin action, stimulation of cell cycle etc.

Phytohormone production is one mechanism by which rhizobacteria can stimulate plant growth, but it is not clear whether the bacteria gain from this mechanism. The hypothesis that microbial-derived cytokinin phytohormones stimulate root exudation of amino acids was tested. The rhizosphere of wheat plants was drenched with the synthetic cytokinin trans-zeatin or inoculated with Bacillus subtilis IB-22 (which produces zeatin type cytokinins) or B. subtilis IB-21 (which failed to accumulate cytokinins). Growing plants in a split root system allowed spatial separation of zeatin application or rhizobacterial inoculation to one compartment and analyses of amino acid release from roots (rhizodeposition) into the other compartment (without either microbial inoculation or treatment with exogenous hormone). Supplying B. subtilis IB-22 or zeatin to either the whole root system or half of the roots increased concentrations of amino acids in the soil solution although the magnitude of the increase was greater when whole roots were treated. There was some similarity in amino acid concentrations induced by either bacterial or zeatin treatment. Thus B. subtilis IB-22 increased amino acid rhizodeposition, likely due to its ability to produce cytokinins. Furthermore, B. subtilis strain IB-21, which failed to accumulate cytokinins in culture media, did not significantly affect amino acid concentrations in the wheat rhizosphere. The ability of rhizobacteria to produce cytokinins and thereby stimulate rhizodeposition may be important in enhancing rhizobacterial colonization of the rhizoplane.

The interactions between the plant hormones auxin and cytokinin throughout plant development are complex, and genetic investigations of the interdependency of auxin and cytokinin signaling have been limited. We have characterized the cytokinin sensitivity of the auxin-resistant diageotropica (dgt) mutant of tomato (Lycopersicon esculentum Mill.) in a range of auxin- and cytokinin-regulated responses. Intact, etiolated dgt seedlings showed cross-resistance to cytokinin with respect to root elongation, but cytokinin effects on hypocotyl growth and ethylene synthesis in these seedlings were not impaired by the dgt mutation. Seven-week-old, green wild-type and dgt plants were also equally sensitive to cytokinin with respect to shoot growth and hypocotyl and internode elongation. The effects of cytokinin and the dgt mutation on these processes appeared additive. In tissue culture organ regeneration from dgt hypocotyl explants showed reduced sensitivity to auxin but normal sensitivity to cytokinin, and the effects of cytokinin and the mutation were again additive. However, although callus induction from dgt hypocotyl explants required auxin and cytokinin, dgt calli did not show the typical concentration-dependent stimulation of growth by either auxin or cytokinin observed in wild-type cells. Cross-resistance of the dgt mutant to cytokinin thus was found to be limited to a small subset of auxin- and cytokinin-regulated growth processes affected by the dgt mutation, indicating that auxin and cytokinin regulate plant growth through both shared and separate signaling pathways.

Rhodococcus equi is an aerobic, gram-positive, non-motile pleomorphic bacillus infecting immunocompromised patients. Forty-nine cases of Rhodococcus equi infection have been reported, mainly in patients infected with the human immunodeficiency virus (HIV). A case in which Rhodococcus equi caused...... severe pulmonary infection, the most common presentation, is described. Clinically, patients have symptoms of pneumonia with hemoptysis as a prominent feature. X-ray will often show a cavitating upper-lobe infiltrate, resembling infection with mycobacteria. Rhodococcus equi is easily cultured from blood...... or sputum on standard media, but is frequently regarded as a contaminant. Mortality from Rhodococcus equi pneumonia is high (25%) and early surgical intervention has been recommended. Based on this review, the benefit of surgery seems dubious, whereas good results have been obtained using long...

Excised roots of Bryophyllum calycinum require for growth both auxin and cytokinin. This is demonstrated by the poor growth of 2-mm root tips in a basal medium of mineral salts, sucrose, and vitamins supplemented with either an auxin or a cytokinin, and much better growth when the basal medium is supplemented with both auxin and cytokinin. However, both substances are synthesized by the root, as is demonstrated by the growth of large inocula (dry wt 6-7 mg) through many successive passages in a medium limited to mineral salts, sugar, and vitamins.

Cytokinin plays a critical role in plant growth and development by regulating cell divisions and cell differentiation. Recent studies suggest that cytokinin signaling is presumably mediated by a two-component system analogous to those found in bacteria and fungi, which transduces an external signal via a phosphorelay from the plasma membrane-anchored receptors to downstream effectors andregulators. Moreover, cytokinin signaling is highly interactive with other pathways, and many components of the pathway appear to be functionally redundant. Proper address of these questions will be crucial for our further understanding onthis important network.

The cucumber cotyledon greening bioassay is frequently used for detecting cytokinins. Beneficial modifications of the original technique included using 5-day-old cucumber (Cucumus sativus L., cv. National Pickling) cotyledons treated with combinations of 40 millimolar KCl and various concentrations of cytokinins. A dark incubation period of 20 hours was followed by an exposure to light for 3.5 hours. Under these conditions, extremely low (0.0001 milligram per liter) concentrations of N6-benzyladenine, zeatin, kinetin, or zeatin riboside can be detected. Of the four cytokinins tested, kinetin appeared to be the least active. With these improvements, the assay is 10 times more sensitive than is the previously described cucumber cotyledon greening bioassay for cytokinins. PMID:16662273

The cucumber cotyledon greening bioassay is frequently used for detecting cytokinins. Beneficial modifications of the original technique included using 5-day-old cucumber (Cucumus sativus L., cv. National Pickling) cotyledons treated with combinations of 40 millimolar KCl and various concentrations of cytokinins. A dark incubation period of 20 hours was followed by an exposure to light for 3.5 hours. Under these conditions, extremely low (0.0001 milligram per liter) concentrations of N(6)-benzyladenine, zeatin, kinetin, or zeatin riboside can be detected. Of the four cytokinins tested, kinetin appeared to be the least active. With these improvements, the assay is 10 times more sensitive than is the previously described cucumber cotyledon greening bioassay for cytokinins.

Full Text Available An investigation was carried out on growth substance requirements of tomato callus derived from anthers for culture in vitro. Linsmaier and Skoog (1965 medium was used with various levels of auxins (IAA and NAA and cytokinins (K and BAP. The results show that cytokinin is an absolute requirement for callus growth irrespective of the auxin level. The optimum concentration of auxin in combination with cytokinin was found to be 5 μM of NAA or 25 μM of IAA, with 5 μM of K or BAP. Callus growth on media with NAA and cytokinin was superior to that on IAA, amounting to 6.05 g per piece on medium with 5 μM of NAA and BAP. Tissues grown on this medium have the highest water content. At the onset of culture the tissue is characterized by weak growth and attains its maximal increase in fresh weight after 6 weeks.

One of the proposed mechanisms by which rhizobacteria enhance plant growth is through the production of plant growth regulators. Five plant growth promoting rhizobacterial (PGPR) strains produced the cytokinin dihydrozeatin riboside (DHZR) in pure culture. Cytokinin production by Pseudomonas fluorescens G20-18, a rifampicin-resistant mutant (RIF), and two TnphoA-derived mutants (CNT1, CNT2), with reduced capacity to synthesize cytokinins, was further characterized in pure culture using immunoassay and thin layer chromatography. G20-18 produced higher amounts of three cytokinins, isopentenyl adenosine (IPA), trans-zeatin ribose (ZR), and DHZR than the three mutants during stationary phase. IPA was the major metabolite produced, but the proportion of ZR and DHZR accumulated by CNT1 and CNT2 increased with time. No differences were observed between strain G20-18 and the mutants in the amounts of indole acetic acid synthesized, nor were gibberellins detected in supernatants of any of the strains. Addition of 10(-5) M adenine increased cytokinin production in 96- and 168-h cultures of strain G20-18 by approximately 67%. G20-18 and the mutants CNT1 and CNT2 may be useful for determination of the role of cytokinin production in plant growth promotion by PGPR.

Actinobacteria of the genus Rhodococcus produce trehalolipid biosurfactants with versatile biochemical properties and low toxicity. In recent years, these biosurfactants are increasingly studied as possible biomedical agents with expressed immunological activities. Applications of trehalolipids from Rhodococcus, predominantly cell-bound, in biomedicine are also attractive because their cost drawback could be less significant for high-value products. The review summarizes recent findings in immunomodulatory activities of trehalolipid biosurfactants from nonpathogenic Rhodococcus and related actinobacteria and compares their biomedical potential with well-known immunomodifying properties of trehalose dimycolates from Mycobacterium tuberculosis. Molecular mechanisms of trehalolipid interactions with immunocompetent cells are also discussed.

Rhodococcus equi is a rare pathogen in humans causing infections mostly in immunocompromised hosts. We present the first case of periprosthetic joint infection caused by Rhodococcus equi. An 88-year-old male patient was referred to our clinic with a history of fever and right hip pain. The patient had multiple hip surgeries including total joint arthroplasty and revision for aseptic loosening on the right side. He was immunocompetent, but his additional medical history was remarkable for diabetes mellitus, diabetic nephropathy and stroke with hemiplegia resulting in immobilization. Radiography showed stable components, joint aspirate yielded Rhodococcus equi. Irrigation and debridement was proposed, but the patient refused any surgical intervention. Therefore antibiotic therapy was administered. At the last follow-up the patient is free of complaints but the C-reactive protein level is still elevated. This case illustrates the possible role of Rhodococcus equi in medical device-associated infections. Orv Hetil. 2017; 158(27): 1071-1074.

Full Text Available Six cultivars of radish (Raphanus sativus were tested for their usefulness in radish cytokinin bioassay by the method of Letham (1971. The best cultivar was found to be 'Sopel Lodu' which responds well to both zeatin and 2iP over a wide range of concentrations. The fresh weight of cotyledons increased at most by 71.5% (if treated with zeatin or 101.0% (if treated with 2iP compared to untreated cotyledons. This cultivar is also sensitive to the partially purified cytokinin-like fraction isolated from the pine (Pinus silvestris cambial region. The cultivar 'Sopel Lodu' is therefore proposed to be a suitable plant for cytokinin bioassays.

The soybean callus bioassay was used to determine levels of cytokinin activity in avocado (Persea americana) seeds.In the embryo, levels are high during the early stages of development, but diminish as the fruit grows. The level of cytokinin activity in the endosperm is very high throughout the period that this tissue exists. The seed coats have very high activity levels while the fruit is young, reaching values comparable with those found in the endosperm. The activity level falls as the rate of fruit growth slows down and disappears completely by the time the seed coats shrivel at approximately the same time the fruit reaches "horticultural maturity".

The crystal structures of complexes of M. truncatula nodulin 13 with four cytokinins, trans-zeatin, N{sup 6}-isopentenyladenine, kinetin and N{sup 6}-benzyladenine, show an unusual mode of dimerization of this PR-10-fold plant protein. The cytokinin-binding mode in the internal cavity of the protein is the same in each complex and resembles the pattern found in the cytokinin receptor protein. Nodulation is an extraordinary symbiotic interaction between leguminous plants and nitrogen-fixing bacteria (rhizobia) that assimilate atmospheric nitrogen (in root nodules) and convert it into compounds suitable for the plant host. A class of plant hormones called cytokinins are involved in the nodulation process. In the model legume Medicago truncatula, nodulin 13 (MtN13), which belongs to the pathogenesis-related proteins of class 10 (PR-10), is expressed in the outer cortex of the nodules. In general, PR-10 proteins are small and monomeric and have a characteristic fold with an internal hydrophobic cavity formed between a seven-stranded antiparallel β-sheet and a C-terminal α-helix. Previously, some PR-10 proteins not related to nodulation were found to bind cytokinins such as trans-zeatin. Here, four crystal structures of the MtN13 protein are reported in complexes with several cytokinins, namely trans-zeatin, N{sup 6}-isopentenyladenine, kinetin and N{sup 6}-benzyladenine. All four phytohormones are bound in the hydrophobic cavity in the same manner and have excellent definition in the electron-density maps. The binding of the cytokinins appears to be strong and specific and is reinforced by several hydrogen bonds. Although the binding stoichiometry is 1:1, the complex is actually dimeric, with a cytokinin molecule bound in each subunit. The ligand-binding site in each cavity is formed with the participation of a loop element from the other subunit, which plugs the only entrance to the cavity. Interestingly, a homodimer of MtN13 is also formed in solution, as confirmed

-senescence activities and the production of a cytokinin mimic by what was once considered a necrotrophic pathogen that promotes cell death and senescence challenges the simple view that this pathogen invades its hosts by employing a barrage of lytic enzymes and toxins. Through genome mining, a gene cluster in the F......The fungal pathogen Fusarium pseudograminearum causes important diseases of wheat and barley. During a survey of secondary metabolites produced by this fungus, a novel class of cytokinins, herein termed Fusarium cytokinins, was discovered. Cytokinins are known for their growth promoting and anti....... pseudograminearum genome for the production of Fusarium cytokinins was identified and the biosynthetic pathway established using gene knockouts. The Fusarium cytokinins could activate plant cytokinin signalling, demonstrating their genuine hormone mimicry. In planta analysis of the transcriptional response to one...

The influence of applied plant growth regulators (PGR) on growth, development and yield in cotton (Gossypium hirsutum L. and Gossypium barbadense L.) has been studied for over half a century. Studies of PGR containing cytokinin alone or in combination with gibbererillins applied at the pinhead squ...

Leaf senescence in plants is a regulated process influenced by light as well as phytohormones. In the present study the putative role of the phytohormones cytokinins and gibberellins as mediators for the light signal on leaf senescence in alstroemeria was studied. It was found that low photon fluenc

The arbuscular mycorrhizal (AM) symbiosis is functionally important for the nutrition and growth of most terrestrial plants. Nearly all phytohormones are employed by plants to regulate the symbiosis with AM fungi, but the regulatory role of cytokinin (CK) is not well understood. Here, we used

The arbuscular mycorrhizal (AM) symbiosis is functionally important for the nutrition and growth of most terrestrial plants. Nearly all phytohormones are employed by plants to regulate the symbiosis with AM fungi, but the regulatory role of cytokinin (CK) is not well understood. Here, we used transg

identified redundancy in several biosynthesis families, we found that mutation of LjIpt4 limits nodule numbers. Overexpression of LjIpt3 or LjLog4 alone was insufficient to produce robust formation of spontaneous nodules. In contrast, overexpressing a complete cytokinin biosynthesis pathway leads to large...

...) that translocates to roots and blocks further nodule development. Here we show that, CLE-RS1/2-HAR1 signalling activates the production of shoot-derived cytokinins, which have an SDI-like capacity to systemically suppress nodulation...

Lysozyme is an important and widespread component of the innate immune response that constitutes the first line of defense against bacterial pathogens. The bactericidal effect of this enzyme relies on its capacity to hydrolyze the bacterial cell wall and also on a nonenzymatic mechanism involving its cationic antimicrobial peptide (CAMP) properties, which leads to membrane permeabilization. In this paper, we report our findings on the lysozyme resistance ability of Rhodococcus equi, a pulmonary pathogen of young foals and, more recently, of immunocompromised patients, whose pathogenic capacity is conferred by a large virulence plasmid. Our results show that (i) R. equi can be considered to be moderately resistant to lysozyme, (ii) the activity of lysozyme largely depends on its muramidase action rather than on its CAMP activity, and (iii) the virulence plasmid confers part of its lysozyme resistance capacity to R. equi. This study is the first one to demonstrate the influence of the virulence plasmid on the stress resistance capacity of R. equi and improves our understanding of the mechanisms enabling R. equi to resist the host defenses.

Rhodococcus is an emerging cause of opportunistic infection in immunocompromised patients, most commonly causing cavitary pneumonia. It has rarely been reported as a cause of isolated bacteremia. However, the relationship between bacteremia and central venous catheter is unknown. Between 2002 and 2010, the characteristics and outcomes of seventeen cancer patients with Rhodococcus bacteremia and indwelling central venous catheters were evaluated. Rhodococcus bacteremias were for the most part (94%) central line-associated bloodstream infection (CLABSI). Most of the bacteremia isolates were Rhodococcus equi (82%). Rhodococcus isolates formed heavy microbial biofilm on the surface of polyurethane catheters, which was reduced completely or partially by antimicrobial lock solution. All CLABSI patients had successful response to catheter removal and antimicrobial therapy. Rhodococcus species should be added to the list of biofilm forming organisms in immunocompromised hosts and most of the Rhodococcus bacteremias in cancer patients are central line associated. PMID:22427914

Full Text Available Rhodococcus is an emerging cause of opportunistic infection in immunocompromised patients, most commonly causing cavitary pneumonia. It has rarely been reported as a cause of isolated bacteremia. However, the relationship between bacteremia and central venous catheter is unknown. Between 2002 and 2010, the characteristics and outcomes of seventeen cancer patients with Rhodococcus bacteremia and indwelling central venous catheters were evaluated. Rhodococcus bacteremias were for the most part (94% central line-associated bloodstream infection (CLABSI. Most of the bacteremia isolates were Rhodococcus equi (82%. Rhodococcus isolates formed heavy microbial biofilm on the surface of polyurethane catheters, which was reduced completely or partially by antimicrobial lock solution. All CLABSI patients had successful response to catheter removal and antimicrobial therapy. Rhodococcus species should be added to the list of biofilm forming organisms in immunocompromised hosts and most of the Rhodococcus bacteremias in cancer patients are central line associated.

Cytokinin regulates many aspects of plant development, and in legume crops, this phytohormone is necessary and sufficient for symbiotic nodule organogenesis, allowing them to fix atmospheric nitrogen. To identify direct links between cytokinins and nodule organogenesis, we determined a consensus sequence bound in vitro by a transcription factor (TF) acting in cytokinin signaling, the nodule-enhanced Medicago truncatula Mt RR1 response regulator (RR). Among genes rapidly regulated by cytokinins and containing this so-called RR binding site (RRBS) in their promoters, we found the nodulation-related Type-A RR Mt RR4 and the Nodulation Signaling Pathway 2 (NSP2) TF. Site-directed mutagenesis revealed that RRBS cis-elements in the RR4 and NSP2 promoters are essential for expression during nodule development and for cytokinin induction. Furthermore, a microRNA targeting NSP2 (miR171 h) is also rapidly induced by cytokinins and then shows an expression pattern anticorrelated with NSP2. Other primary targets regulated by cytokinins depending on the Cytokinin Response1 (CRE1) receptor were a cytokinin oxidase/dehydrogenase (CKX1) and a basic Helix-Loop-Helix TF (bHLH476). RNA interference constructs as well as insertion of a Tnt1 retrotransposon in the bHLH gene led to reduced nodulation. Hence, we identified two TFs, NSP2 and bHLH476, as direct cytokinin targets acting at the convergence of phytohormonal and symbiotic cues. PMID:23023168

Rhodococcus equi is a well-recognized pathogen in veterinary medicine that can also affect immuno-compromised human subjects. The most common clinical features in humans include necrotizing pneumonia with subacute pulmonary disease, progressive cough, chest pain and fever. We report a case of a 49-year-old kidney transplant patient who developed a Rhodococcus equi infection characterized by multiple abscesses of the soft tissues and muscles without any respiratory manifestation. Combining specific antibiotic therapy and surgical management of the abscesses without immunosuppression discontinuation led to a complete recovery of both patient and graft.

The rate-limiting step of cytokinin biosynthesis in Arabidopsis thaliana Heynh. is catalyzed by ATP/ADP isopentenyltransferases, A. thaliana IsoPentenyl Transferase (AtIPT)1, and AtIPT4, and by their homologs AtIPT3, AtIPT5, AtIPT6, AtIPT7, and AtIPT8. To understand the dynamics of cytokinins in plant development, we comprehensively analyzed the expression of isopentenyltransferase genes of Arabidopsis. Examination of their mRNA levels and the expression patterns of the beta-glucuronidase (GUS) gene fused to the regulatory sequence of each AtIPT gene revealed a specific expression pattern of each gene. The predominant expression patterns were as follows: AtIPT1::GUS, xylem precursor cell files in the root tip, leaf axils, ovules, and immature seeds; AtIPT3::GUS, phloem tissues; AtIPT4::GUS and AtIPT8::GUS, immature seeds with highest expression in the chalazal endosperm (CZE); AtIPT5::GUS, root primordia, columella root caps, upper part of young inflorescences, and fruit abscission zones; AtIPT7::GUS, endodermis of the root elongation zone, trichomes on young leaves, and some pollen tubes. AtIPT1, AtIPT3, AtIPT5, and AtIPT7 were downregulated by cytokinins within 4 h. AtIPT5 and AtIPT7 was upregulated by auxin within 4 h in roots. AtIPT3 was upregulated within 1 h after an application of nitrate to mineral-starved Arabidopsis plants. The upregulation by nitrate did not require de novo protein synthesis. We also examined the expression of two genes for tRNA isopentenyltransferases, AtIPT2 and AtIPT9, which can also be involved in cytokinin biosynthesis. They were expressed ubiquitously, with highest expression in proliferating tissues. These findings are discussed in relation to the role of cytokinins in plant development.

Full Text Available The phytohormones cytokinin and auxin are essential for the control of diverse aspects of cell proliferation and differentiation processes in plants. Although both phytohormones have been suggested to play key roles in the regulation of root nodule development, only recently, significant progress has been made in the elucidation of the molecular genetic basis of cytokinin action in the model leguminous species, Lotus japonicus and Medicago truncatula. Identification and functional analyses of the putative cytokinin receptors LOTUS HISTIDINE KINASE 1 and M. truncatula CYTOKININ RESPONSE 1 have brought a greater understanding of how activation of cytokinin signaling is crucial to the initiation of nodule primordia. Recent studies have also started to shed light on the roles of auxin in the regulation of nodule development. Here, we review the history and recent progress of research into the roles of cytokinin and auxin, and their possible interactions, in nodule development.

Full Text Available Imogen Johns Department of Clinical Sciences and Services, Royal Veterinary College, North Mymms, UK Abstract: Rhodococcus equi, a gram-positive facultative intracellular bacterial pathogen, is the most important cause of pneumonia in foals aged 3 weeks to 5 months. The disease occurs worldwide, resulting in significant morbidity and mortality on endemically affected farms. Foals appear to become infected early in life, but clinical signs are typically delayed until 1–3 months of age because of the insidious nature of the disease. Although pneumonia is the most common clinical manifestation, up to 74% of foals may concurrently have extrapulmonary disorders, including both extrapulmonary infections (abdominal abscessation, colitis, osteomyelitis and immune-mediated disorders (nonseptic synovitis, uveitis. Diagnosis is based on the combination of clinical signs and abnormalities on hematologic screening and thoracic imaging in an appropriately aged foal and is confirmed by bacteriologic culture of the organism. Management of R. equi infections, in particular on farms with endemic disease, combines appropriate treatment of affected foals with preventative measures targeted at preventing infection and identifying foals before the development of severe disease. The combination of rifampin and a macrolide antimicrobial is recommended for treatment, as the combination is synergistic, reaches high intracellular concentrations, and should minimize the development of antimicrobial resistance. The prognosis for survival for foals with R. equi pneumonia is good, especially in foals mildly or subclinically affected, as is the prognosis for future athletic performance. Screening for early identification before the development of clinical signs has been advocated on endemically affected farms, although the most appropriate method, the timing of screening, and the selection of foals requiring treatment have yet to be determined. Recent evidence suggests that

Full Text Available BACKGROUND: When applied to a nutrition solution or agar media, the non-substituted aromatic cytokinins caused thickening and shortening of the primary root, had an inhibitory effect on lateral root branching, and even showed some negative effects on development of the aerial part at as low as a 10 nanomolar concentration. Novel analogues of aromatic cytokinins ranking among topolins substituted on N9-atom of adenine by tetrahydropyranyl or 4-chlorobutyl group have been prepared and tested in standardized cytokinin bioassays [1]. Those showing comparable activities with N(6-benzylaminopurine were further tested in planta. METHODOLOGY/PRINCIPAL FINDINGS: The main aim of the study was to explain molecular mechanism of function of novel cytokinin derivatives on plant development. Precise quantification of cytokinin content and profiling of genes involved in cytokinin metabolism and perception in treated plants revealed several aspects of different action of m-methoxytopolin base and its substituted derivative on plant development. In contrast to standard cytokinins, N9- tetrahydropyranyl derivative of m-topolin and its methoxy-counterpart showed the negative effects on root development only at three orders of magnitude higher concentrations. Moreover, the methoxy-derivative demonstrates a positive effect on lateral root branching and leaf emerging in a nanomolar range of concentrations, in comparison with untreated plants. CONCLUSIONS/SIGNIFICANCE: Tetrahydropyranyl substitution at N9-position of cytokinin purine ring significantly enhances acropetal transport of a given cytokinins. Together with the methoxy-substitution, impedes accumulation of non-active cytokinin glucoside forms in roots, allows gradual release of the active base, and has a significant effect on the distribution and amount of endogenous isoprenoid cytokinins in different plant tissues. The utilization of novel aromatic cytokinin derivatives can distinctively improve expected

or sputum on standard media, but is frequently regarded as a contaminant. Mortality from Rhodococcus equi pneumonia is high (25%) and early surgical intervention has been recommended. Based on this review, the benefit of surgery seems dubious, whereas good results have been obtained using long...

Putrescine oxidase from Rhodococcus erythropolis (PuO) is a flavin-containing amine oxidase from the monoamine oxidase family that performs oxidative deamination of aliphatic diamines. In this study we report pre-steady-state kinetic analyses of the enzyme with the use of single-and double-mixing st

Bile acids are highly abundant steroids with important functions in vertebrate digestion. Their catabolism by bacteria is an important component of the carbon cycle, contributes to gut ecology, and has potential commercial applications. We found that Rhodococcus jostii RHA1 grows well on cholate, as

Putrescine oxidase from Rhodococcus erythropolis (PuO) is a flavin-containing amine oxidase from the monoamine oxidase family that performs oxidative deamination of aliphatic diamines. In this study we report pre-steady-state kinetic analyses of the enzyme with the use of single-and double-mixing

Full Text Available A large range of microorganisms can associate with plants, resulting in neutral, friendly or hostile interactions. The ability of plants to recognize compatible and incompatible microorganisms and to limit or promote their colonization is therefore crucial for their survival. Elaborated communication networks determine the degree of association between the host plant and the invading microorganism. Central to these regulations of plant microbe interactions, phytohormones modulate microorganism plant associations and coordinate cellular and metabolic responses associated to the progression of microorganisms across different plant tissues. We review here hormonal regulations, focusing on auxin and cytokinin phytohormones, involved in the interactions between plant roots and soil microorganisms, including bacteria and fungi associations, either beneficial (symbiotic or detrimental (pathogenic. The aim is to highlight similarities and differences in cytokinin/auxin functions amongst various compatible versus incompatible associations.

Objective:To determine the effects of different cytokinins at various concentrations on in vitro shoot multiplication of an important medicinal plant. Methods: Nodal explants (1.5-2.0 cm) of Sophora tonkinensis were used. Multiple shoots were induced from nodal explants cultured on the Murashige and Skoog (MS) medium supplemented with 0.0, 0.5, 1.0, 2.0, 4.0, 8.0, or 16.0 µmol 2-isopentyladenine (2iP), N6 benzyladenine, kinetin or thiadiazuron. Results: Among the four investigated cytokinins, 2iP showed the best response for shoot multiplication. Maximum shoot induction (75%) was achieved on the MS medium supplemented with 2.0 µmol 2iP, with a mean number of 5.0 shoots per explant. In comparison to other cytokinins tried, 2iP showed the highest shoot elongation with a mean shoot length of 4.8 cm. Root initiation was observed within 15 d within the transfer of shoots onto the MS basal medium, and the rooting percentage was 100%with a mean number of 5.4 roots per shoot and root length of 6.2 cm over a period of 4 weeks. The healthy plants, hardened and transferred to a greenhouse for proper acclimatization, exhibited 100%survival. Conclusions:It can be summarized that 2iP is the optimal plant growth regulator for Sophora multiplication.

Legumes establish symbiotic associations with nitrogen-fixing bacteria (rhizobia) in root nodules to obtain nitrogen. Legumes control nodule number through long-distance communication between roots and shoots, maintaining the proper symbiotic balance. Rhizobial infection triggers the production of mobile CLE-RS1/2 peptides in Lotus japonicus roots; the perception of the signal by receptor kinase HAR1 in shoots presumably induces the production of an unidentified shoot-derived inhibitor (SDI) that translocates to roots and blocks further nodule development. Here we show that, CLE-RS1/2-HAR1 signalling activates the production of shoot-derived cytokinins, which have an SDI-like capacity to systemically suppress nodulation. In addition, we show that LjIPT3 is involved in nodulation-related cytokinin production in shoots. The expression of LjIPT3 is activated in an HAR1-dependent manner. We further demonstrate shoot-to-root long-distance transport of cytokinin in L. japonicus seedlings. These findings add essential components to our understanding of how legumes control nodulation to balance nutritional requirements and energy status.

Cytokinins have been implicated in normal plant growth and development. These bioactive molecules are essential for cell production and expansion in higher plants. Carrot is an Apiaceae vegetable with great value and undergoes significant size changes over the process of plant growth. However, cytokinin accumulation and its potential roles in carrot growth have not been elucidated. To address this problem, carrot plants at five stages were collected, and morphological and anatomical characteristics and expression profiles of cytokinin-related genes were determined. During carrot growth and development, cytokinin levels were the highest at the second stage in the roots, whereas relatively stable levels were observed in the petioles and leaves. DcCYP735A2 showed high expression at stage 2 in the roots, which may contribute largely to the higher cytokinin level at this stage. However, expression of most metabolic genes did not follow a pattern similar to that of cytokinin accumulation, indicating that cytokinin biosynthesis was regulated through a complex network. Genes involved in cytokinin signal perception and transduction were also integrated to normal plant growth and development. The results from the present work suggested that cytokinins may regulate plant growth in a stage-dependent manner. Our work would shed novel insights into cytokinin accumulation and its potential roles during carrot growth. Further studies regarding carrot cytokinins may be achieved by modification of the genes involved in cytokinin biosynthesis, inactivation, and perception.

Full Text Available Cytokinins have been implicated in normal plant growth and development. These bioactive molecules are essential for cell production and expansion in higher plants. Carrot is an Apiaceae vegetable with great value and undergoes significant size changes over the process of plant growth. However, cytokinin accumulation and its potential roles in carrot growth have not been elucidated. To address this problem, carrot plants at five stages were collected, and morphological and anatomical characteristics and expression profiles of cytokinin-related genes were determined. During carrot growth and development, cytokinin levels were the highest at the second stage in the roots, whereas relatively stable levels were observed in the petioles and leaves. DcCYP735A2 showed high expression at stage 2 in the roots, which may contribute largely to the higher cytokinin level at this stage. However, expression of most metabolic genes did not follow a pattern similar to that of cytokinin accumulation, indicating that cytokinin biosynthesis was regulated through a complex network. Genes involved in cytokinin signal perception and transduction were also integrated to normal plant growth and development. The results from the present work suggested that cytokinins may regulate plant growth in a stage-dependent manner. Our work would shed novel insights into cytokinin accumulation and its potential roles during carrot growth. Further studies regarding carrot cytokinins may be achieved by modification of the genes involved in cytokinin biosynthesis, inactivation, and perception.

Full Text Available Cytokinins in plants are crucial for numerous biological processes, including seed germination, cell division and differentiation, floral initiation and adaptation to abiotic stresses. The salt stress can promote reactive oxygen species (ROS production in plants which are highly toxic and ultimately results in oxidative stress. However, the correlation between endogenous cytokinin production and ROS homeostasis in responding to salt stress is poorly understood. In this study, we analyzed the correlation of overexpressing the cytokinin biosynthetic gene AtIPT8 (adenosine phosphate-isopentenyl transferase 8 and the response of salt stress in Arabidopsis. Overproduction of cytokinins, which was resulted by the inducible overexpression of AtIPT8, significantly inhibited the primary root growth and true leaf emergence, especially under the conditions of exogenous salt, glucose and mannitol treatments. Upon cytokinin overproduction, the salt stress resistance was declined, and resulted in less survival rates and chlorophyll content. Interestingly, ROS production was obviously increased with the salt treatment, accompanied by endogenously overproduced cytokinins. The activities of CAT and SOD, which are responsible for scavenging ROS, were also affected. Transcription profiling revealed that the differential expressions of ROS-producing and scavenging related genes, the photosynthesis-related genes and stress responsive genes were existed in transgenic plants of overproducing cytokinins. Our results suggested that broken in the homeostasis of cytokinins in plant cells could modulate the salt stress responses through a ROS-mediated regulation in Arabidopsis.

Cytokinins promote cell division and chloroplast development in tissue culture. We previously isolated two mutants of Arabidopsis thaliana, ckh1 (cytokinin-hypersensitive 1) and ckh2, which produce rapidly growing green calli in response to lower levels of cytokinins than those found in the wild type. Here we report that the product of the CKH2 gene is PICKLE, a protein resembling the CHD3 class of SWI/SNF chromatin remodeling factors. We also show that inhibition of histone deacetylase by trichostatin A (TSA) partially substituted for cytokinins, but not for auxin, in the promotion of callus growth, indicating that chromatin remodeling and histone deacetylation are intimately related to cytokinin-induced callus growth. A microarray experiment revealed that either the ckh1 mutation or the ckh2 mutation caused hypersensitivity to cytokinins in terms of gene expression, especially of photosynthesis-related genes. The ckh1 and ckh2 mutations up-regulated nuclear-encoded genes, but not plastid-encoded genes, whereas TSA deregulated both nuclear- and plastid-encoded genes. The ckh1 ckh2 double mutant showed synergistic phenotypes: the callus grew with a green color independently of exogenous cytokinins. A yeast two-hybrid experiment showed protein interaction between CKH1/EER4/AtTAF12b and CKH2/PKL. These results suggest that CKH1/EER4/AtTAF12b and CKH2/PKL may act together on cytokinin-regulated genes.

Cytokinins, a class of phytohormones, are adenine derivatives common to many different organisms. In plants, these play a crucial role as regulators of plant development and the reaction to abiotic and biotic stress. Key enzymes in the cytokinin synthesis and degradation in modern land plants are the isopentyl transferases and the cytokinin dehydrogenases, respectively. Their encoding genes have been probably introduced into the plant lineage during the primary endosymbiosis. To shed light on the evolution of these proteins, the genes homologous to plant adenylate isopentenyl transferase and cytokinin dehydrogenase were amplified from the genomic DNA of cyanobacterium Nostoc sp. PCC 7120 and expressed in Escherichia coli. The putative isopentenyl transferase was shown to be functional in a biochemical assay. In contrast, no enzymatic activity was detected for the putative cytokinin dehydrogenase, even though the principal domains necessary for its function are present. Several mutant variants, in which conserved amino acids in land plant cytokinin dehydrogenases had been restored, were inactive. A combination of experimental data with phylogenetic analysis indicates that adenylate-type isopentenyl transferases might have evolved several times independently. While the Nostoc genome contains a gene coding for protein with characteristics of cytokinin dehydrogenase, the organism is not able to break down cytokinins in the way shown for land plants.

Cytokinins, a class of phytohormones, are adenine derivatives common to many different organisms. In plants, these play a crucial role as regulators of plant development and the reaction to abiotic and biotic stress. Key enzymes in the cytokinin synthesis and degradation in modern land plants are

Legume rhizobium symbiosis is initiated upon perception of bacterial secreted lipo-chitooligosaccharides (LCOs). Perception of these signals by the plant initiates a signaling cascade that leads to nodule formation. Several studies have implicated a function for cytokinin in this process. However, whether cytokinin accumulation and subsequent signaling are an integral part of rhizobium LCO signaling remains elusive. Here, we show that cytokinin signaling is required for the majority of transcriptional changes induced by rhizobium LCOs. In addition, we demonstrate that several cytokinins accumulate in the root susceptible zone 3 h after rhizobium LCO application, including the biologically most active cytokinins, trans-zeatin and isopentenyl adenine. These responses are dependent on calcium- and calmodulin-dependent protein kinase (CCaMK), a key protein in rhizobial LCO-induced signaling. Analysis of the ethylene-insensitive Mtein2/Mtsickle mutant showed that LCO-induced cytokinin accumulation is negatively regulated by ethylene. Together with transcriptional induction of ethylene biosynthesis genes, it suggests a feedback loop negatively regulating LCO signaling and subsequent cytokinin accumulation. We argue that cytokinin accumulation is a key step in the pathway leading to nodule organogenesis and that this is tightly controlled by feedback loops.

CKX (cytokinin dehydrogenase) is a flavoprotein that cleaves cytokinins to adenine and the corresponding side-chain aldehyde using a quinone-type electron acceptor. In the present study, reactions of maize (Zea mays) CKX with five different substrates (N6-isopentenyladenine, trans-zeatin, kinetin, p

CKX (cytokinin dehydrogenase) is a flavoprotein that cleaves cytokinins to adenine and the corresponding side-chain aldehyde using a quinone-type electron acceptor. In the present study, reactions of maize (Zea mays) CKX with five different substrates (N6-isopentenyladenine, trans-zeatin, kinetin,

Cytokinins in plants are crucial for numerous biological processes, including seed germination, cell division and differentiation, floral initiation and adaptation to abiotic stresses. The salt stress can promote reactive oxygen species (ROS) production in plants which are highly toxic and ultimately results in oxidative stress. However, the correlation between endogenous cytokinin production and ROS homeostasis in responding to salt stress is poorly understood. In this study, we analyzed the correlation of overexpressing the cytokinin biosynthetic gene AtIPT8 (adenosine phosphate-isopentenyl transferase 8) and the response of salt stress in Arabidopsis. Overproduction of cytokinins, which was resulted by the inducible overexpression of AtIPT8, significantly inhibited the primary root growth and true leaf emergence, especially under the conditions of exogenous salt, glucose and mannitol treatments. Upon cytokinin overproduction, the salt stress resistance was declined, and resulted in less survival rates and chlorophyll content. Interestingly, ROS production was obviously increased with the salt treatment, accompanied by endogenously overproduced cytokinins. The activities of catalase (CAT) and superoxide dismutase (SOD), which are responsible for scavenging ROS, were also affected. Transcription profiling revealed that the differential expressions of ROS-producing and scavenging related genes, the photosynthesis-related genes and stress responsive genes were existed in transgenic plants of overproducing cytokinins. Our results suggested that broken in the homeostasis of cytokinins in plant cells could modulate the salt stress responses through a ROS-mediated regulation in Arabidopsis.

Full Text Available We studied the effects of various cytokinins and urea derivatives on germination of aged seeds of in Lotus corniculatus L. The following substances were applied: N6-isoprenoid cytokinins (isopentenyl adenine and zeatin, adenine sulfate, N6-aromatic cytokinins (kinetin, benzyladenine and their N9-ribosides, N-benzyl-9-(2- tetrahydropyranyladenine, and urea derivatives (diphenylurea, thidiazuron, and chloro-pyridyl phenylurea. With the exception of adenine sulfate, all cytokinins increased the percentage of seed germination up to twofold, depending on their kind and concentration. It is concluded that cytokinins may be among the missing factors in aged seeds of L. corniculatus contributing to the implementation of their full germination potential. They could be used to improve germination of both freshly harvested and aged seed samples, if necessary. .

For decades researchers have been targeting prevention of Rhodococcus equi (Rhodococcus hoagui/Prescottella equi) by vaccination and the horse breeding industry has supported the ongoing efforts by researchers to develop a safe and cost effective vaccine to prevent disease in foals. Traditional vaccines including live, killed and attenuated (physical and chemical) vaccines have proved to be ineffective and more modern molecular-based vaccines including the DNA plasmid, genetically attenuated and subunit vaccines have provided inadequate protection of foals. Newer, bacterial vector vaccines have recently shown promise for R. equi in the mouse model. This article describes the findings of key research in R. equi vaccine development and looks at alternative methods that may potentially be utilised.

Cytokinins are plant hormones that have, among many other functions, senescence-modulatory effects in plant tissue. This is evident not only from biochemical data, but is vividly illustrated in the "green island" phenotype in plant leaves caused by cytokinins released for example by leaf mining insects or microbial pathogens. It is beyond doubt that, in addition to their roles in plants, cytokinins also provoke physiological and developmental effects in animals. It is hypothesized that the recently much discussed modification of plant metabolism by insects and associated microbes via cytokinin signals has a counterpart in direct cytokinin signalling that interferes with the animals' hormonal systems and impacts their population dynamics.

Full Text Available There is world wide concern about the liberation of hydrocarbons in the environment, both from industrial activities and from accidental spills of oil and oilrelated compounds. Biosurfactants, which are natural emulsifiers of hydrocarbons, are produced by some bacteria, fungi and yeast. They are polymers, totally or partially extracellular, with an amphipathyc structure, which allows them to form micelles that accumulate at the interface between liquids of different polarities such as water and oil. This process is based upon the ability of biosurfactants to reduce surface tension, blocking the formation of hydrogen bridges and certain hydrophilic and hydrophobic interactions. The ability of biosurfactant production by five strains of Rhodococcus isolated from oil prospecting sites was evaluated. Surface tension measurement and emulsifying index were used to quantify biosurfactant production. The influence of environmental conditions was also investigated - pH, temperature, medium composition, and type of carbon source - on cell growth and biosurfactant production. Strain AC 239 was shown to be a potential producer, attaining 63% of emulsifying index for a Diesel-water binary system. It could be used, either directly on oil spills in contained environments, or for the biotechnological production of biosurfactant.

Quantitative aspects of fecal Rhodococcus (Corynebacterium) equi in newborn foals for 12 weeks after birth were investigated on two horse breeding farms. R. equi was found in the feces of foals during week 1 of life. The greatest numbers of R. equi were present in the feces of foals during the first 8 weeks of their lives, which coincides with the age when foals are most liable to be exposed to R. equi.

Full Text Available Rhodococcus equi has emerged as an opportunistic pathogen associated with pulmonary, invasive or systemic infections in immunocompromised patients. We report the identification of 51 R. equi isolates found in sputum samples of 546 individuals suspected to have pulmonary tuberculosis in two Public Health Hospital Units in Brazil. The epidemiology of R. equi infection as well as the phenotypic identification and drug susceptibility profile of isolates are described in this paper.

We report a comparative study of 29 representative genomes of the animal pathogen Rhodococcus equi The analyses showed that R. equi is genetically homogeneous and clonal, with a large core genome accounting for ≈80% of an isolates' gene content. An open pangenome, even distribution of accessory genes among the isolates, and absence of significant core-genome recombination, indicated that gene gain/loss is a main driver of R. equi genome evolution. Traits previously predicted to be important in R. equi physiology, virulence and niche adaptation were part of the core genome. This included the lack of a phosphoenolpyruvate:carbohydrate transport system (PTS), unique among the rhodococci except for the closely related Rhodococcus defluvii, reflecting selective PTS gene loss in the R. equi-R. defluvii sublineage. Thought to be asaccharolytic, rbsCB and glcP non-PTS sugar permease homologues were identified in the core genome and, albeit inefficiently, R. equi utilized their putative substrates, ribose and (irregularly) glucose. There was no correlation between R. equi whole-genome phylogeny and host or geographical source, with evidence of global spread of genomovars. The distribution of host-associated virulence plasmid types was consistent with the exchange of the plasmids (and corresponding host shifts) across the R. equi population, and human infection being zoonotically acquired. Phylogenomic analyses demonstrated that R. equi occupies a central position in the Rhodococcus phylogeny, not supporting the recently proposed transfer of the species to a new genus.

Full Text Available Rhodococcus equi is a common cause of pneumonia in foals and has extensive clinical, economic and possibly zoonotic consequences. This bacterium survives well in the environment and may be considered as normal flora of adult horses. Certain strains of this bacterium are extremely virulent in foals, and early identification and intervention is crucial for prognosis. Rhodococcus equi is endemic in many parts of the world and occasionally isolated in Israel. This study was designed to evaluate R. equi seroprevalence in adult horses in Israel to indirectly indicate the potential level of exposure of susceptible foals. Sera were collected from 144 horses during spring 2011 and from 293 horses during fall 2014, and the presence of antibodies against virulent R. equi was detected by enzyme-linked immunosorbent assay. Equine seroprevalence of R. equi was found to be 7.6% in 2011 and 5.1% in 2014. Only one farm had seropositive horses in 2011, whereas several farms had seropositive horses in 2014. No significant risk factors for seropositivity were found. Rhodococcus equi appears to be endemic in Israel. This is the first survey of R. equi in Israel that provides information on the epidemiology of this important bacterium.

Rhodococcus equi is a common cause of pneumonia in foals and has extensive clinical, economic and possibly zoonotic consequences. This bacterium survives well in the environment and may be considered as normal flora of adult horses. Certain strains of this bacterium are extremely virulent in foals, and early identification and intervention is crucial for prognosis. Rhodococcus equi is endemic in many parts of the world and occasionally isolated in Israel. This study was designed to evaluate R. equi seroprevalence in adult horses in Israel to indirectly indicate the potential level of exposure of susceptible foals. Sera were collected from 144 horses during spring 2011 and from 293 horses during fall 2014, and the presence of antibodies against virulent R. equi was detected by enzyme-linked immunosorbent assay. Equine seroprevalence of R. equi was found to be 7.6% in 2011 and 5.1% in 2014. Only one farm had seropositive horses in 2011, whereas several farms had seropositive horses in 2014. No significant risk factors for seropositivity were found. Rhodococcus equi appears to be endemic in Israel. This is the first survey of R. equi in Israel that provides information on the epidemiology of this important bacterium.

Gram-positive bacteria of the genus Rhodococcus catabolize p-hydroxybenzoate (PHB) through the initial formation of 3,4-dihydroxybenzoate. High levels of p-hydroxybenzoate hydroxylase (PHBH) activity are induced in six different Rhodococcus species when these strains are grown on PHB as sole carbon

Flagellates are very important predators on bacteria in soil. Because of their high growth rates, flagellate populations respond rapidly to changes in bacterial numbers. Previous results indicate that actinobacteria are generally less suitable than proteobacteria as food for flagellates. In this ...

Micropropagation is used for commercial purposes worldwide, but the capacity to undergo somatic organogenesis and plant regeneration varies greatly among species. The plant hormones auxin and cytokinin are critical for plant regeneration in tissue culture, with cytokinin playing an instrumental role in shoot organogenesis. Type-B response regulators govern the transcriptional output in response to cytokinin and are required for plant regeneration. In our paper published in Plant Physiology, we explored the functional redundancy among the 11 type-B Arabidopsis response regulators (ARRs). Interestingly, we discovered that the enhanced expression of one family member, ARR10, induced hypersensitivity to cytokinin in multiple assays, including callus greening and shoot induction of explants. Here we 1) discuss the hormone dependence for in vitro plant regeneration, 2) how manipulation of the cytokinin response has been used to enhance plant regeneration, and 3) the potential of the ARR10 transgene as a tool to increase the regeneration capacity of agriculturally important crop plants. The efficacy of ARR10 for enhancing plant regeneration likely arises from its ability to transcriptionally regulate key cytokinin responsive genes combined with an enhanced protein stability of ARR10 compared with other type-B ARRs. By increasing the capacity of key tissues and cell types to respond to cytokinin, ARR10, or other type-B response regulators with similar properties, could be used as a tool to combat the recalcitrance of some crop species to tissue culture techniques.

As sessile organisms, plants must sense environmental conditions and adjust their growth and development processes accordingly, through adaptive responses regulated by various internal factors, including hormones. A key environmental factor is temperature, but temperature-sensing mechanisms are not fully understood despite intense research. We investigated proteomic responses to temperature shocks (15 min cold or heat treatments) with and without exogenous applications of cytokinin in Arabidopsis. Image and mass spectrometric analysis of the two-dimensionally separated proteins detected 139 differentially regulated spots, in which 148 proteins were identified, most of which have not been previously linked to temperature perception. More than 70% of the temperature-shock response proteins were modulated by cytokinin, mostly in a similar manner as heat shock. Data mining of previous transcriptomic datasets supported extensive interactions between temperature and cytokinin signalling. The biological significance of this finding was tested by assaying an independent growth response of Arabidopsis seedlings to heat stress: hypocotyl elongation. This response was strongly inhibited in mutants with deficiencies in cytokinin signalling or endogenous cytokinin levels. Thus, cytokinins may directly participate in heat signalling in plants. Finally, large proportions of both temperature-shock and cytokinin responsive proteomes co-localize to the chloroplast, which might therefore host a substantial proportion of the temperature response machinery.

While the molecular basis for cytokinin action is quite well understood in flowering plants, little is known about the cytokinin signal transduction in early diverging land plants. The genome of the bryophyte Physcomitrella patens (Hedw.) B.S. encodes three classical cytokinin receptors, the CHASE domain-containing histidine kinases, CHK1, CHK2, and CHK3. In a complementation assay with protoplasts of receptor-deficient Arabidopsis thaliana as well as in cytokinin binding assays, we found evidence that CHK1 and CHK2 receptors can function in cytokinin perception. Using gene targeting, we generated a collection of CHK knockout mutants comprising single (Δchk1, Δchk2, Δchk3), double (Δchk1,2, Δchk1,3, Δchk2,3), and triple (Δchk1,2,3) mutants. Mutants were characterized for their cytokinin response and differentiation capacities. While the wild type did not grow on high doses of cytokinin (1 µM benzyladenine), the Δchk1,2,3 mutant exhibited normal protonema growth. Bud induction assays showed that all three cytokinin receptors contribute to the triggering of budding, albeit to different extents. Furthermore, while the triple mutant showed no response in this bioassay, the remaining mutants displayed budding responses in a diverse manner to different types and concentrations of cytokinins. Determination of cytokinin levels in mutants showed no drastic changes for any of the cytokinins; thus, in contrast to Arabidopsis, revealing only small impacts of cytokinin signaling on homeostasis. In summary, our study provides a first insight into the molecular action of cytokinin in an early diverging land plant and demonstrates that CHK receptors play an essential role in bud induction and gametophore development.

The expression of a new acylamidase gene from R. erythropolis 37 was studied in Rhodococcus erythropolis strains. This acylamidase, as a result of its unique substrate specificity, can hydrolyse N-substituted amides (4'-nitroacetanilide, N-isopropylacrylamide, N'N-dimethylaminopropylacrylamide). A new expression system based on the use of the promoter region of nitrilhydratase genes from R. rhodochrous M8 was created to achieve constitutive synthesis of acylamidase in R. erythropolis cells. A fourfold improvement in the acylamidase activity of recombinant R. erythropolis cells as compared with the parent wild-type strain was obtained through the use of the new expression system.

Full Text Available There have been reports of Rhodococcus equi infections in goats in Australia, America and India. In this study, R. equi was isolated from an inflamed vertebra in a Boer goat in South Africa. At autopsy, there was a purulent inflammatory reaction in the 1st cervical vertebra. Histopathologically, a neutrophilic infiltration was encountered in the bone. Aerobic culture of swabs collected from the abscesses yielded R. equi in pure culture that was identified on biochemical tests. R. equi has become important as the cause of an opportunistic infection in people suffering from HIV.

Full Text Available Rhodococcus equi , previously known as Corynebacterium equi, is one of the most important causes of zoonotic infection in grazing animals. Increased cases of human infection with R. equi have been reported especially in immunocompromised patients. Infection in immunocompetent patients is extremely rare. We report a case of R. equi bacteremia in a 26-day-old immunocompetent infant with recurrent swellings on different parts of the body. To the best of our knowledge, this is the first ever report of R. equi bacteremia from an immunocompetent patient from Northern India.

When grown in the absence of light, the det1 mutants of Arabidopsis thaliana develop characteristics of light-grown plants by morphological, cellular, and molecular criteria. Further, in light-grown plants, mutations in the DET1 gene affect cell-type-specific expression of light-regulated genes and the chloroplast developmental program. Here we show that the addition of exogenously added cytokinins (either 2-isopentenyl adenine, kinetin, or benzyladenine) to the growth medium of dark-germinated wild-type seedlings results in seedlings that resemble det1 mutants, instead of having the normal etiolated morphology. Like det1 mutants, these dark-grown seedlings now contain chloroplasts and have high levels of expression of genes that are normally light''-regulated. These results suggest an important role for cytokinins during greening of Arabidopsis, and may implicate cytokinin levels or an increased sensitivity to cytokinins as explanations for some of the observed phenotypes of det1 mutants.

Leachate extracts from senescent Gingko biloba leaf compost, separated by paper chromatography, and analyzed for biological activity using the soybean callus bioassay indicated that cytokinin-like compounds which co-eluted with zeatin and/or dihydrozeatin are released from decomposing leaves. Both leachate and leaf compost extracts obtained in the 5th month of composting were fractionated using HPLC. A cytokinin-like compound, which co-eluted with iso-pentenyladenosine was detected.

A novel nicotine-degrading bacterium, strain Y22, was isolated and identified as Rhodococcus sp. Y22 based on its 16S rDNA sequence and morphological and biochemical features. The isolate could utilize nicotine as the sole source of carbon and nitrogen. Nicotine (1.0g/L) was degraded by Rhodococcus sp. Y22 within 52h at 28 degrees C and pH 7.0. Preparation of resting cells from nicotine-induced cultures was found to rapidly and efficiently degrade nicotine from solutions as well as from tobacco leaves. Therefore, Rhodococcus sp. Y22 has the potential to degrade nicotine during tobacco leave processing.

Vernalization is required for floral initiation in Dendrobium. Interestingly, those beneficial effects can also be achieved by exogenous cytokinin application in greenhouses. Thus, an as yet unknown crosstalk/interaction may exist between vernalization and cytokinin signaling pathways. In this study, we showed, by de novo transcriptome assembly using RNA-seq data from both vegetative and reproductive tissue samples, that some floral transition-related genes-DnVRN1, FT, SOC1, LFY and AP1-were differentially expressed in low-temperature-challenged (LT) or thidiazuron (TDZ)-treated plants, compared to those mock-treated (CK). Both LT and TDZ upregulated SOC1, LFY and AP1, while the upregulation of DnVRN1 and FT was only LT-induced. We further found that LT promoted the upregulation of some key cytokinin signaling regulators, including several cytokinin biosynthesis-related genes and type-B response regulator (RR)-encoding genes, and that both LT and TDZ triggered the significant upregulation of some marker genes in the gibberellin (GA) signaling pathway, indicating an important low temperature-cytokinin-GA axis in flowering. Our data thus have revealed a cytokinin-GA signal network underlying vernalization, providing a novel insight into further investigation of the molecular mechanism of floral initiation in Dendrobium.

Vernalization is required for floral initiation in Dendrobium. Interestingly, those beneficial effects can also be achieved by exogenous cytokinin application in greenhouses. Thus, an as yet unknown crosstalk/interaction may exist between vernalization and cytokinin signaling pathways. In this study, we showed, by de novo transcriptome assembly using RNA-seq data from both vegetative and reproductive tissue samples, that some floral transition-related genes—DnVRN1, FT, SOC1, LFY and AP1—were differentially expressed in low-temperature-challenged (LT) or thidiazuron (TDZ)-treated plants, compared to those mock-treated (CK). Both LT and TDZ upregulated SOC1, LFY and AP1, while the upregulation of DnVRN1 and FT was only LT-induced. We further found that LT promoted the upregulation of some key cytokinin signaling regulators, including several cytokinin biosynthesis-related genes and type-B response regulator (RR)-encoding genes, and that both LT and TDZ triggered the significant upregulation of some marker genes in the gibberellin (GA) signaling pathway, indicating an important low temperature-cytokinin-GA axis in flowering. Our data thus have revealed a cytokinin-GA signal network underlying vernalization, providing a novel insight into further investigation of the molecular mechanism of floral initiation in Dendrobium. PMID:28361995

Silicate minerals are dominant soil components. Thus, plant roots are constantly exposed to silicic acid. High silicon intake, enabled by root silicon transporters, correlates with increased tolerance to many biotic and abiotic stresses. However, the underlying protection mechanisms are largely unknown. Here, we tested the hypothesis that silicon interacts with the plant hormones, and specifically, that silicic acid intake increases cytokinin biosynthesis. The reaction of sorghum (Sorghum bicolor) and Arabidopsis plants, modified to absorb high versus low amounts of silicon, to dark-induced senescence was monitored, by quantifying expression levels of genes along the senescence pathway and measuring tissue cytokinin levels. In both species, detached leaves with high silicon content senesced more slowly than leaves that were not exposed to silicic acid. Expression levels of genes along the senescence pathway suggested increased cytokinin biosynthesis with silicon exposure. Mass spectrometry measurements of cytokinin suggested a positive correlation between silicon exposure and active cytokinin concentrations. Our results indicate a similar reaction to silicon treatment in distantly related plants, proposing a general function of silicon as a stress reliever, acting via increased cytokinin biosynthesis.

The cytokinin activity has been isolated and identified from extracts of immature mango (Mangifera indica L.) seeds. The structures of zeatin, zeatin riboside, and N(6)-(Delta(2)-isopentenyl)adenine riboside were confirmed on the basis of their chromatographic behavior and mass spectra of trimethylsilyl derivatives. Both trans and cis isomers of zeatin and zeatin riboside were also identified by the retention times of high performance liquid chromatography. In addition, an unidentified compound appeared to be a cytokinin glucoside.The concentration of cytokinins in the panicle and pulp of mango reached a maximum 5 to 10 days after full bloom and decreased rapidly thereafter. The cytokinin level in the seed remained high until the 28th day after full bloom. The quantity of cytokinins in pulp per fruit increased from the 10th day after full bloom, the maximum being attained around the 50th day after full bloom. Similarly, the amount of cytokinins per seed increased from the 10th day after full bloom, reaching a peak on the 40th day and decreasing gradually thereafter.A high percentage of fruit set in mango was persistently maintained by supplying 6-benzylaminopurine (1.5 x 10(3) micromolar) onto the panicle at the anthesis stage and by supplying gibberellic acid (7.2 x 10(2) micromolar) and naphthalene acetamide (3.1 x 10 micromolar) at the young fruit stage.

An unusual case of cavitary Rhodococcus equi pneumonia with endobronchial granulomas in congenital HIV infection is presented. The clinical features and radiological manifestations of pulmonary R. equi infection are discussed.

Reproductive development in the Christmas rose (Helleborus niger L.) differs from that in commonly investigated model plants in two important aspects: (i) the perianth develops a photosynthetic system, after fertilization, and persists until seed ripening; and (ii) the ripe seed contains an immature embryo which continues to mature off the mother plant. The possible roles of cytokinins in these processes are investigated here by analysing extracts of the perianth and the carpels/maturing fruit prepared during anthesis and four stages of post-floral development. trans-Zeatin, dihydrozeatin, N6-(Delta2-isopentenyl)adenine, and their ribosides were identified by tandem mass spectrometry. Single ion monitoring in the presence of deuterated internal standards demonstrated the additional presence of the corresponding riboside-5'-monophosphates, O-glucosides, and 9-glucosides, and afforded quantitative data on the whole set of endogenous cytokinins. Fruit cytokinins were mostly localized in the seeds. Their overall concentrations increased dramatically during early seed development and remained high for 6-8 weeks, until shortly before seed ripening (the last time point covered in this work). Overall cytokinin levels in the perianth did not change markedly in the period covered, but the level of N6-(Delta2-isopentenyl)adenine-type cytokinins appeared to increase slightly and transiently during the greening phase. The perianths of unpollinated or depistillated flowers, which survived, but did not pass through the complete greening process, contained significantly less cytokinins than observed in fruit-bearing flowers. This suggests that perianth greening requires defined cytokinin levels and supports the role of the developing fruit in their maintenance.

.... To genetically dissect the cytokinin pathway, we have used a functional screen to identify Arabidopsis gain-of-function mutations that enable shoot formation in the absence of exogenous cytokinins...

Full Text Available Abstract This study sought to determine whether an increase in resistance of Rhodococcus equi to the antibiotics rifampin and erythromycin occurred over a 10-year period. This was carried out by the use of E test strips for rifampin and erythromycin to determine the MIC (minimum inhibitory concentration values of Rhodococcus equi to this combination of antibiotics. The findings of this study indicated that there was an increase in resistance of Rhodococcus equi to rifampin and erythromycin over the 10-year period. The MIC for rifampin increased from 0.081 μg/ml in 1996 to 0.187 μg/ml in 2006 and from 0.258 μg/ml for erythromycin during the years prior to 2000 to 0.583 μg/ml in 2006. This finding suggests that there may be a problem in the treatment of Rhodococcus equi infections in foals in the future, particularly as the number of drugs available for treatment of Rhodococcus equi infection is limited because of the intracellular capabilities of this bacterium. Antibiotics used in its treatment have to be able to penetrate the polysaccharide cell wall of Rhodococcus equi as well as the alveolar macrophages in which the bacterium is capable of surviving.

In this study, coaggregation interactions between Rhodococcus and Acinetobacter strains isolated from food-processing surfaces were characterized. Rhodococcus sp. strain MF3727 formed intrageneric coaggregates with Rhodococcus sp. strain MF3803 and intergeneric coaggregates with 2 strains of Acinetobacter calcoaceticus (MF3293, MF3627). Stronger coaggregation between A. calcoaceticus MF3727 and Rhodococcus sp. MF3293 was observed after growth in batch culture at 30 °C than at 20 °C, after growth in tryptic soy broth than in liquid R2A medium, and between cells in exponential and early stationary phases than cells in late stationary phase. The coaggregation ability of Rhodococcus sp. MF3727 was maintained even after heat and Proteinase K treatment, suggesting its ability to coaggregate was protein independent whereas the coaggregation determinants of the other strains involved proteinaceous cell-surface-associated polymers. Coaggregation was stable at pH 5-9. The mechanisms of coaggregation among Acinetobacter and Rhodococcus strains bare similarity to those displayed by coaggregating bacteria of oral and freshwater origin, with respect to binding between proteinaceous and nonproteinaceous determinants and the effect of environmental factors on coaggregation. Coaggregation may contribute to biofilm formation on industrial food surfaces, protecting bacteria against cleaning and disinfection.

In this paper comparative genome and phenotype microarray analyses of Rhodococcus sp. BCP1 and Rhodococcus opacus R7 were performed. Rhodococcus sp. BCP1 was selected for its ability to grow on short-chain n-alkanes and R. opacus R7 was isolated for its ability to grow on naphthalene and on o-xylene. Results of genome comparison, including BCP1, R7, along with other Rhodococcus reference strains, showed that at least 30% of the genome of each strain presented unique sequences and only 50% of the predicted proteome was shared. To associate genomic features with metabolic capabilities of BCP1 and R7 strains, hundreds of different growth conditions were tested through Phenotype Microarray, by using Biolog plates and plates manually prepared with additional xenobiotic compounds. Around one-third of the surveyed carbon sources was utilized by both strains although R7 generally showed higher metabolic activity values compared to BCP1. Moreover, R7 showed broader range of nitrogen and sulphur sources. Phenotype Microarray data were combined with genomic analysis to genetically support the metabolic features of the two strains. The genome analysis allowed to identify some gene clusters involved in the metabolism of the main tested xenobiotic compounds. Results show that R7 contains multiple genes for the degradation of a large set of aromatic and PAHs compounds, while a lower variability in terms of genes predicted to be involved in aromatic degradation was found in BCP1. This genetic feature can be related to the strong genetic pressure exerted by the two different environment from which the two strains were isolated. According to this, in the BCP1 genome the smo gene cluster involved in the short-chain n-alkanes degradation, is included in one of the unique regions and it is not conserved in the Rhodococcus strains compared in this work. Data obtained underline the great potential of these two Rhodococcus spp. strains for biodegradation and environmental decontamination

In this paper comparative genome and phenotype microarray analyses of Rhodococcus sp. BCP1 and Rhodococcus opacus R7 were performed. Rhodococcus sp. BCP1 was selected for its ability to grow on short-chain n-alkanes and R. opacus R7 was isolated for its ability to grow on naphthalene and on o-xylene. Results of genome comparison, including BCP1, R7, along with other Rhodococcus reference strains, showed that at least 30% of the genome of each strain presented unique sequences and only 50% of the predicted proteome was shared. To associate genomic features with metabolic capabilities of BCP1 and R7 strains, hundreds of different growth conditions were tested through Phenotype Microarray, by using Biolog plates and plates manually prepared with additional xenobiotic compounds. Around one-third of the surveyed carbon sources was utilized by both strains although R7 generally showed higher metabolic activity values compared to BCP1. Moreover, R7 showed broader range of nitrogen and sulphur sources. Phenotype Microarray data were combined with genomic analysis to genetically support the metabolic features of the two strains. The genome analysis allowed to identify some gene clusters involved in the metabolism of the main tested xenobiotic compounds. Results show that R7 contains multiple genes for the degradation of a large set of aromatic and PAHs compounds, while a lower variability in terms of genes predicted to be involved in aromatic degradation was found in BCP1. This genetic feature can be related to the strong genetic pressure exerted by the two different environment from which the two strains were isolated. According to this, in the BCP1 genome the smo gene cluster involved in the short-chain n-alkanes degradation, is included in one of the unique regions and it is not conserved in the Rhodococcus strains compared in this work. Data obtained underline the great potential of these two Rhodococcus spp. strains for biodegradation and environmental decontamination

Full Text Available The majority of infections caused by R. equi occur in hosts with some degree of cell-mediated immunodeficiency. Immunocompetent individuals are infrequently affected and usually present with localized disease. Infections of the skin or related structures are uncommon and are usually related to environmental contamination. The microbiology laboratory plays a key role in the identification of the organism since it may be mistaken for common skin flora. We describe a 31 year-old woman without medical problems who presented nine weeks after breast reduction with right breast cellulitis and purulent drainage from the surgical wound. She underwent incision and drainage, and cultures of the wound yielded Rhodococcus equi. The patient completed six weeks of antimicrobial therapy with moxifloxacin and rifampin with complete resolution.

Full Text Available Rhodococcus equi is a respiratory pathogen which primarily infects foals and is endemic on farms around the world with 50% mortality and 80% morbidity in affected foals. Unless detected early and treated appropriately the disease can be fatal. Currently, there is no vaccine available to prevent this disease. For decades researchers have endeavoured to develop an effective vaccine to no avail. In this study a novel human adenoviral vector vaccine for R. equi was developed and tested in the mouse model. This vaccine generated a strong antibody and cytokine response and clearance of R. equi was demonstrated following challenge. These results show that this vaccine could potentially be developed further for use as a vaccine to prevent R. equi disease in foals.

Rhodococcus equi is a respiratory pathogen which primarily infects foals and is endemic on farms around the world with 50% mortality and 80% morbidity in affected foals. Unless detected early and treated appropriately the disease can be fatal. Currently, there is no vaccine available to prevent this disease. For decades researchers have endeavoured to develop an effective vaccine to no avail. In this study a novel human adenoviral vector vaccine for R. equi was developed and tested in the mouse model. This vaccine generated a strong antibody and cytokine response and clearance of R. equi was demonstrated following challenge. These results show that this vaccine could potentially be developed further for use as a vaccine to prevent R. equi disease in foals.

Cytokinins (CKs) are one of the main regulators of in vitro growth and development and might affect the developmental state and function of the photosynthetic apparatus of in vitro shoots. Effects of different cytokinin regimes including different types of aromatic cytokinins, such as benzyl-adenine, benzyl-adenine riboside and 3-hydroxy-benzyladenine alone or in combination were studied on the capacity of the photosynthetic apparatus and the pigment content of in vitro apple leaves after 3 weeks of culture. We found that the type of cytokinins affected both chlorophyll a and b contents and its ratio. Chlorophyll content of in vitro apple leaves was the highest when benzyl-adenine was applied as a single source of cytokinin in the medium (1846-2176 μg/1g fresh weight (FW) of the leaf). Increasing the concentration of benzyl-adenine riboside significantly decreased the chlorophyll content of the leaves (from 1923 to 1183 μg/1g FW). The highest chl a/chl b ratio was detected after application of meta-topolin (TOP) at concentrations of 2.0 and 6.0 μM (2.706 and 2.804). Chlorophyll fluorescence was measured both in dark-adapted (Fv/Fm test) and in light-adapted leaf samples (Yield test; Y(II)). The maximum quantum yield and efficiency of leaves depended on the cytokinin source of the medium varied between 0.683 and 0.861 (Fv/Fm) indicating a well-developed and functional photosynthetic apparatus. Our results indicate that the type and concentration of aromatic cytokinins applied in the medium affect the chlorophyll content of the leaves in in vitro apple shoots. Performance of the photosynthetic apparatus measured by chlorophyll fluorescence in the leaves was also modified by the cytokinin supply. This is the first ever study on the relationship between the cytokinin supply and the functionability of photosystem II in plant tissue culture and our findings might help to increase plantlet survival after transfer to ex vitro conditions.

Full Text Available The study was done with the objective of tannic acid degradation by tannase from the Rhodococcus NCIM 2891 which is actually a desulfurizing bacteria. Rhodococcus NCIM 2891cultivated in medium containing 0.1% Tannic acid, produced tannase which showed maximum activity after 24 h. The enzyme was purified by DEAE cellulose ion exchange chromatography and was shown to be dimeric in nature it has two subunits as Tannase I and Tannase II, having specific activities of 0.23 U/mg of protein and 0.295 U/mg of protein, respectively. Both the enzyme showed optimum activity at pH 6 and 30ºC. The Km of both enzymes was 0.034 and 0.040 mM respectively and Vmax of both enzymes were found to be 40 and 45 U/mL, respectively. SDS-PAGE analysis of purified proteins fraction revealed that their molecular weights are 60 and 62 kDa. Tannase I was completely inhibited by 10 mM Hg2+, Cu2+, Fe3+, Co2+ and Tannase I and II both was completely inhibited by1 mM Hg2+ alone. The DNA damage- protecting activity of tannic acid and product obtained after tannic acid hydrolysis was assessed bringing about the DNA damage with H2O2 + UV exposure in absence of the hydrolyzed products of tannic acid. The experiment was carried out by using agarose gel electrophoresis to analyze DNA damage in presence of H2O2 + UV. However, there was no damage of DNA by H2O2 + UV exposure in presence of the hydrolysis products.

Changes in primary metabolism of lettuce, Lactuca sativa L. (cv. Cobham Green), induced by compatible interaction with the biotrophic oomycete pathogen Bremia lactucae Regel (race BL 16), under two intensities of illumination in the presence and absence of exogenous cytokinins were studied by chlorophyll fluorescence imaging. Thirteen days post-inoculation leaf discs infected by B. lactucae exhibited impairments of photosynthesis associated with biotrophic infections, including: reductions in photosynthetic pigment contents and the maximum quantum yield of photosystem II photochemistry (F(V)/F(M)), inhibition of electron transport (Phi(PSII)) and increased non-photochemical chlorophyll fluorescence quenching (NPQ). Detected changes in photosynthetic parameters correlated with the leaf area colonized by the pathogen's intercellular hyphae. Applications of two cytokinins, benzylaminopurine and meta-topolin, previously shown to suppress B. lactucae sporulation if applied 24 h prior to inoculation at a concentration of 200 microM, retarded the pathogen's asexual reproduction with no apparent negative effects on the host's photosynthetic apparatus. However, long-lasting treatment of healthy tissues with this high concentration of exogenous cytokinin led to effects parallel to pathogenesis: reductions in photosynthetic pigment contents accompanied by inhibition of photosystem II photochemistry and electron transport. These effects of both prolonged exposure to cytokinins and the pathogenesis were weaker in discs exposed to the lower photosynthetic photon flux density. The role of cytokinins in plant-biotrophic pathogen interactions and their potential as disease control agents are discussed.

Full Text Available ABSTRACT Plant growth regulators can influence fruit yield and quality. This study aimed to evaluate the effect of cytokinin and gibberelin on the agronomic and physicochemical characteristics of banana fruits cv. ‘Prata’ (Musa spp. AAB, according to the formation period and position in the bunch. The experiment was conducted in a completely randomized 2 x 5 factorial design, two periods of bunch development (summer and winter, five treatments and ten replicates. To study the effect of position in the bunch, split plot was adopted, considering in the plot, 2 x 5 factorial and in subplots, hand 1, hand 4 and last hand. Treatments consisted of 2 pulverizations with water, 150 mg L-1 cytokinin, 200 mg L-1 of gibberellic acid, 100 mg L-1 of cytokinin plus 200 mg L-1 of gibberellic acid and 200 mg L-1 of cytokinin plus 200 mg L-1 of gibberellic acid, applied from the fourth to the last hand of the bunch. Cytokinin and gibberellin, alone or associated, regardless of formation period and position, did not affect the size and physicochemical characteristics of fruits, only delayed the bunch harvest.

In order to investigate the interaction of the plant hormones ethylene, abscisic acid (ABA) and cytokinin in seed germination and early seedling development, we studied germination in ethylene-related mutants of Arabidopsis. Mutations in the genes etr1 and ein2, which reduce ethylene responses, showed increased dormancy and a delay in germination in comparison with wild type. Mutations in etr1, ein2 and ein6 also resulted in increased sensitivity to ABA with respect to inhibition of germination. Conversely, mutations in ctr1 and eto3, which lead to an increased ethylene response and overproduction of ethylene, respectively, decreased sensitivity to ABA during germination. Increased ABA sensitivity was also effected in wild type seeds by the presence during germination of AgNO3, an inhibitor of ethylene action. The addition of the cytokinin N-6 benzyl adenine (BA) reversed the increased sensitivity of ethylene-resistant mutants to ABA. The action of cytokinin in reversing increased ABA sensitivity of ethylene-resistant mutants also suggests that at least part of the action of cytokinin in promoting germination is independent of its role in stimulating ethylene production. These observations further extend the evidence in support of interaction between ethylene, ABA and cytokinin signalling in controlling seed germination and early seedling development in Arabidopsis.

"Lonely guy" (LOG) has been identified as a cytokinin-producing enzyme in plants and plant-interacting fungi. The gene product of Cg2612 from the soil-dwelling bacterium Corynebacterium glutamicum was annotated as an LDC. However, the facts that C. glutamicum lacks an LDC and Cg2612 has high amino acid similarity with LOG proteins suggest that Cg2612 is possibly an LOG protein. To investigate the function of Cg2612, we determined its crystal structure at a resolution of 2.3 Å. Cg2612 functions as a dimer and shows an overall structure similar to other known LOGs, such as LOGs from Arabidopsis thaliana (AtLOG), Claviceps purpurea (CpLOG), and Mycobacterium marinum (MmLOG). Cg2612 also contains a "PGGXGTXXE" motif that contributes to the formation of an active site similar to other LOGs. Moreover, biochemical studies on Cg2612 revealed that the protein has phosphoribohydrolase activity but not LDC activity. Based on these structural and biochemical studies, we propose that Cg2612 is not an LDC family enzyme, but instead belongs to the LOG family. In addition, the prenyl-binding site of Cg2612 (CgLOG) comprised residues identical to those seen in AtLOG and CpLOG, albeit dissimilar to those in MmLOG. The work provides structural and functional implications for LOG-like proteins from other microorganisms.

Identification of mutants with impairments in auxin biosynthesis and dynamics by forward genetic screening is hindered by the complexity, redundancy and necessity of the pathways involved. Furthermore, although a few auxin-deficient mutants have been recently identified by screening for altered responses to shade, ethylene, N-1-naphthylphthalamic acid (NPA) or cytokinin (CK), there is still a lack of robust markers for systematically isolating such mutants. We hypothesized that a potentially suitable phenotypic marker is root curling induced by CK, as observed in the auxin biosynthesis mutant CK-induced root curling 1 / tryptophan aminotransferase of Arabidopsis 1 (ckrc1/taa1). Phenotypic observations, genetic analyses and biochemical complementation tests of Arabidopsis seedlings displaying the trait in large-scale genetic screens showed that it can facilitate isolation of mutants with perturbations in auxin biosynthesis, transport and signaling. However, unlike transport/signaling mutants, the curled (or wavy) root phenotypes of auxin-deficient mutants were significantly induced by CKs and could be rescued by exogenous auxins. Mutants allelic to several known auxin biosynthesis mutants were re-isolated, but several new classes of auxin-deficient mutants were also isolated. The findings show that CK-induced root curling provides an effective marker for discovering genes involved in auxin biosynthesis or homeostasis.

The presence of arsenic in soil and water is a constant threat to plant growth in many regions of the world. Phytohormones act in the integration of growth control and stress response, but their role in plant responses to arsenic remains to be elucidated. Here, we show that arsenate [As(V)], the most prevalent arsenic chemical species in nature, causes severe depletion of endogenous cytokinins (CKs) in the model plant Arabidopsis (Arabidopsis thaliana). We found that CK signaling mutants and transgenic plants with reduced endogenous CK levels showed an As(V)-tolerant phenotype. Our data indicate that in CK-depleted plants exposed to As(V), transcript levels of As(V)/phosphate-transporters were similar or even higher than in wild-type plants. In contrast, CK depletion provoked the coordinated activation of As(V) tolerance mechanisms, leading to the accumulation of thiol compounds such as phytochelatins and glutathione, which are essential for arsenic sequestration. Transgenic CK-deficient Arabidopsis and tobacco lines show a marked increase in arsenic accumulation. Our findings indicate that CK is an important regulatory factor in plant adaptation to arsenic stress.

Legumes develop different types of lateral organs from their primary root, lateral roots and nodules, the latter depending on a symbiotic interaction with Sinorhizobium meliloti. Phytohormones have been shown to function in the control of these organogeneses. However, related signaling pathways have not been identified in legumes. We cloned and characterized the expression of Medicago truncatula genes encoding members of cytokinin signaling pathways. RNA interference of the cytokinin receptor homolog Cytokinin Response1 (Mt CRE1) led to cytokinin-insensitive roots, which showed an increased number of lateral roots and a strong reduction in nodulation. Both the progression of S. meliloti infection and nodule primordia formation were affected. We also identified two cytokinin signaling response regulator genes, Mt RR1 and Mt RR4, which are induced early during the symbiotic interaction. Induction of these genes by S. meliloti infection is altered in mutants affected in the Nod factor signaling pathway; conversely, cytokinin regulation of the early nodulin Nodule Inception1 (Mt NIN) depends on Mt CRE1. Hence, cytokinin signaling mediated by a single receptor, Mt CRE1, leads to an opposite control of symbiotic nodule and lateral root organogenesis. Mt NIN, Mt RR1, and Mt RR4 define a common pathway activated during early S. meliloti interaction, allowing crosstalk between plant cytokinins and bacterial Nod factors signals.

Plant beneficial microbes mediate biocontrol of diseases by interfering with pathogens or via strengthening the host. Although phytohormones, including cytokinins, are known to regulate plant development and physiology as well as plant immunity, their production by microorganisms has not been considered as a biocontrol mechanism. Here we identify the ability of Pseudomonas fluorescens G20-18 to efficiently control P. syringae infection in Arabidopsis, allowing maintenance of tissue integrity and ultimately biomass yield. Microbial cytokinin production was identified as a key determinant for this biocontrol effect on the hemibiotrophic bacterial pathogen. While cytokinin-deficient loss-of-function mutants of G20-18 exhibit impaired biocontrol, functional complementation with cytokinin biosynthetic genes restores cytokinin-mediated biocontrol, which is correlated with differential cytokinin levels in planta. Arabidopsis mutant analyses revealed the necessity of functional plant cytokinin perception and salicylic acid-dependent defence signalling for this biocontrol mechanism. These results demonstrate microbial cytokinin production as a novel microbe-based, hormone-mediated concept of biocontrol. This mechanism provides a basis to potentially develop novel, integrated plant protection strategies combining promotion of growth, a favourable physiological status and activation of fine-tuned direct defence and abiotic stress resilience.

Cytokinins represent a group of plant hormones that have been shown to be essential for plant growth and development. A recent large-scale phylogenetic analysis of components of the cytokinin signal transduction pathway revealed, among other findings, the existence of a second, previously unknown

The recessive ckh1 (cytokinin hypersensitive 1) mutant of Arabidopsis thaliana shows hypersensitivity to cytokinins, which promote proliferation and greening of calli. The CKH1 gene encodes a protein resembling TAF12 (TATA BOX BINDING PROTEIN ASSOCIATED FACTOR 12), which is a component of transcription factor IID (TFIID)- and histone acetyltransferase-containing complexes in yeast and animals. Microarray analyses revealed that a substantially greater number of genes responded to a low level of cytokinins in the ckh1 mutant than in the wild type. However, expression of cytokinin primary response genes was not significantly affected by the ckh1 mutation. These results suggest that the CKH1 protein regulates a set of genes involved in late signaling processes governing a range of cytokinin responses, including cell proliferation and differentiation.

Endogenous cytokinin (CK) levels of in vitro-cultured and greenhouse-acclimatized 'Williams' bananas treated with six aromatic CKs were quantified using UPLC-MS/MS. The underground parts had higher endogenous CK levels than the aerial parts. Control plantlets had more isoprenoid CKs while the aromatic-type CKs were predominant in all other regenerants. Following acclimatization of the control and 10 μM CK regenerants, there was a rapid decline in both isoprenoid and aromatic CK in the greenhouse-grown plants. Apart from the control and 6-(3-Methoxybenzylamino)-9-tetrahydropyran-2-ylpurine (MemTTHP) treatment with higher level of isoprenoid CK, aromatic CK remain the predominant CK-type across all CK treatments. The most abundant CK forms were meta-topolin (mT) and benzyladenine (BA) in the micropropagated and acclimatized plants, respectively. Micropropagated plantlets had cis-Zeatin (cZ) as the major isoprenoid CK-type which was in turn replaced by isopentenyladenine (iP) upon acclimatization. On a structural and functional basis, 9-glucoside, a deactivation/detoxicification product was the most abundant and mainly located in the underground parts (micropropagation and acclimatization). The results establish the wide variation in metabolic products of the tested aromatic CKs during micropropagation and acclimatization. The findings are discussed with the possible physiological roles of the various CK constituents on the growth and development of banana plants.

The relationship of air pollution factors to observed forest decline can be investigated from different viewpoints incorporating physiological and biochemical changes. A hormone imbalance can be the result of growth disturbances, as a direct or indirect effect of air pollution. To prove this hypothesis, within an air pollution exclusion experiment in Open Top Chambers at the Edelmannshof site, the variations in the content of different cytokinins were analyzed in Norway spruce needles at various times during annual growth. The first approach adapted the cytokinin extraction and purification method, which is classically used in the laboratory. A second approach presented a one-year time course of the cytokinin content in one-year-old Norway spruce needles. (orig./MG)

The embryonic axis plays an essential role in the mobilization of the main reserves of the cotyledons of seeds of Cicer arietinum L. cv Castellana. This control by the axis of the metabolism of the storage products of the cotyledons largely takes place through the cytokinins, which are transported from the embryonic axis to the cotyledons where the mobilization of reserves begins. The principal regulatory role of the endogenous cytokinins concerns the metabolism of carbohydrates and proteins; there is less influence on lipid metabolism. However, each cytokinin seems to have a different role in the mobilization processes. The glucosides, glucosyl zeatin riboside, and glucosyl zeatin act only as storage forms of the hormones. Zeatin riboside affects mainly the mobilization of carbohydrates and has less effect on protein mobilization. Zeatin regulates both the mobilization of carbohydrates and that of proteins and is more marked in the latter case. PMID:16667549

Various urea-derived herbicides and different cytokinin analogues were used to determine their effects on callusing response and shoot regenerating capacity of alfalfa (Medicago sativa L.) and Coleus (Coleus forskohlii Briq.). The herbicides monuron and diuron evoked profuse callusing response from Coleus leaf segments and alfalfa petiole explants on Murashige and Skoog medium. Shoot regeneration by monuron (2.0 mg/l) showed a maximum of 3 multiple shoots both in alfalfa and Coleus with a frequency of 92% and 75%, respectively. Whereas diuron (0.5 mg/l) showed a high frequency of shoot regeneration (89%)with a mean number of 5 shoots in alfalfa, in C.forskohlii, the frequency of regeneration was 90%with a mean number of 6 shoots. Diuron with two chloride groups in the phenyl ring showed significantly higher cytokinin-like activity than single chloride substitution monuron. This study demonstrates the potential use of monuron and diuron as cytokinins in plant tissue culture.

Various urea-derived herbicides and different cytokinin analogues were used to determine their effects on callusing response and shoot regenerating capacity of alfalfa (Medicago sativa L.) and Coleus (Coleus forskohlii Briq.). The herbicides monuron and diuron evoked profuse callusing response from Coleus leaf segments and alfalfa petiole explants on Murashige and Skoog medium. Shoot regeneration by monuron (2.0 mg/l) showed a maximum of 3 multiple shoots both in alfalfa and Coleus with a frequency of 92% and 75%, respectively. Whereas diuron (0.5 mg/l) showed a high frequency of shoot regeneration (89%) with a mean number of 5 shoots in alfalfa, in C. forskohlii, the frequency of regeneration was 90% with a mean number of 6 shoots. Diuron with two chloride groups in the phenyl ring showed significantly higher cytokinin-like activity than single chloride substitution monuron. This study demonstrates the potential use of monuron and diuron as cytokinins in plant tissue culture.

Hormonal regulation of root development is a long known phenomenon. In the past decades, the molecular mechanisms of individual hormonal pathways and their impact on root development have been studied. Recent genetic and molecular studies suggest importance of interactions of the individual hormonal pathways and their components. In our paper1 we show impact of endogenous cytokinin on the root architecture and its interaction with auxin in Arabidopsis thaliana. In this addendum we discuss our results in the light of significant recent papers that deal with cytokinin-auxin interactions and we point out spatiotemporal specificity of these interactions in the root development. PMID:19649199

Endogenous levels of cytokinin nucleotides in an anise cell culture were determined during proembryonal, as well as embryonal development. In both cultures the maximum level of isopentenyladenine nucleotides was found during the first four days of incubation which correlated with the beginning of logarithmic growth (embryonal: 8 ng g(-1) tresh weight; proembryonal: 17.4 ng g(-1) fresh weight). The concentration of zeatin nucleotides remained constant at a very low level. The present data and those of Ernst et al. (1984) and Ernst and Oesterhelt (1984) are concerned in ascribing a major role to cytokinins in cell division, but not in embryo differentiation.

Summary The long-distance signaling network allowing a plant to properly develop its root system is crucial to optimize root foraging in areas where nutrients are available. Cytokinin is an essential element of the systemic signaling network leading to the enhancement of lateral root proliferation in areas where nitrate is available. Here, we explore more precisely: (i) which particular traits of lateral root growth (density and length of emerged lateral roots) are the targets of systemic signaling in a context of heterogeneous nitrate supply; and (i ) if the systemic signaling depends only on cytokinin or on a combination of several signalings.

Based on a nomenclatural point of view, the name Rhodococcus equi is associated, as required by the Bacteriological Code, with a defined position, rank and circumscription. A search of the literature indicates that the name Rhodococcus equi (Magnusson 1923) Goodfellow and Alderson 1977 has also been shown to be a synonym of Corynebacterium equi Magnusson 1923, Corynebacterium hoagii (Morse 1912) Eberson 1918 and Nocardia restricta (Turfitt 1944) McClung 1974. Application of the rules of the Bacteriological Code together with the currently inferred taxonomic concept associated with the species bearing the name Rhodococcus equi indicates that this is not the correct name of this taxon and the use of that name in the context of a circumscription that includes the type strain of the species Corynebacterium hoagii is contrary to the Rules of the Code.

Rhodococcus (R). equi, a recognized pathogen in horses, is emerging as a human opportunistic pathogen, especially in immunocompromized people. It affects also New World camelids, but there are no reports of R. equi infection in Old World camelids yet. Four cases of disseminated R. equi infection in adult breeding dromedaries occurred at one camel farm near Dubai within 16 months of each other. At necropsy the lungs were diffusely consolidated with large caseous areas. Histology revealed severe suppurative to necrotising pneumonia with multiple encapsulated abscesses. Immunohistochemistry enabled the detection of 15- to 17-kDa antigens (VapA) of R. equi in the lung sections. High numbers of R. equi were isolated from the lung lesions as well as from liver, spleen and mediastinal lymph nodes, indicative of septicaemia. The isolated strains were PCR-positive for the specific virulence plasmid (VapA-Gen) of R. equi, indicating virulent strains and containing an 85-kb type I plasmid. This is the first report of disseminated R. equi infection in Old World camelids. Since adult camels in general do not suffer from bacterial caused pneumonia (except tuberculosis), this is a new emerging disease for camels.

Putrescine oxidase from Rhodococcus erythropolis (PuO) is a flavin-containing amine oxidase from the monoamine oxidase family that performs oxidative deamination of aliphatic diamines. In this study we report pre-steady-state kinetic analyses of the enzyme with the use of single- and double-mixing stopped-flow spectroscopy and putrescine as a substrate. During the fast and irreversible reductive half-reaction no radical intermediates were observed, suggesting a direct hydride transfer from the substrate to the FAD. The rate constant of flavin reoxidation depends on the ligand binding; when the imine product was bound to the enzyme the rate constant was higher than with free enzyme species. Similar results were obtained with product-mimicking ligands and this indicates that a ternary complex is formed during catalysis. The obtained kinetic data were used together with steady-state rate equations derived for ping-pong, ordered sequential and bifurcated mechanisms to explore which mechanism is operative. The integrated analysis revealed that PuO employs a bifurcated mechanism due to comparable rate constants of product release from the reduced enzyme and reoxidation of the reduced enzyme-product complex.

Full Text Available Aim: The aim was to determine the occurrence of Rhodococcus equi in equines and their environment in Jammu (R.S. Pura, Katra, molecular characterization and to determine the antibiotic resistance pattern of R. equi. Materials and Methods: A total of 96 nasopharyngeal swab samples were collected from equines. The organism was isolated on Columbia nalidixic acid agar containing 5% sheep blood as well as on sheep blood agar and was later confirmed by cultural characteristics and biochemical tests. Molecular detection of R. equi isolates was done by 16S rRNA gene amplification followed by virulence associated protein A (Vap A gene amplification. Antibiogram was performed against five antibiotics, viz., amoxicillin, penicillin G, streptomycin, rifampicin, and methicillin. Results: During the study, 9 R. equi isolates were identified on the basis of cultural and biochemical tests. In the polymerase chain reaction based detection, 3 among the 9 rhodococcal isolates were positive for species-specific 16S rRNA gene and revealed amplicon of 450 bp for confirmation of 16S rRNA gene. None of the sample was found positive for Vap A gene. In antibiogram, R. equi isolates were found sensitive for amoxicillin, while some isolates were also found resistant to the most conventional antibiotic penicillin G. Conclusion: From this study, it was concluded that R. equi infection is prevalent in equines in Jammu region of India and the indiscriminate use of the antibiotics is leading toward the development of resistant strains of R. equi.

Full Text Available INTRODUCTION: Rhodococcus equi is an opportunistic pathogen, causing rhodococcosis, a condition that can be confused with tuberculosis. Often, without identifying M. tuberculosis, physicians initiate empiric treatment for tuberculosis. R. equi and M. tuberculosis have different susceptibility to drugs. Identification of R. equi is based on a variety of phenotypic, chromatographic, and genotypic characteristics. OBJECTIVE: This study aimed to characterize bacterial isolates from sputum samples suggestive of R. equi. METHODS: The phenotypic identification included biochemical assays; thin-layer chromatography (TLC and polymerase chain reaction (PCR were used for genotypic identification. RESULTS: Among 78 Gram-positive and partially acid-fast bacilli isolated from the sputum of tuberculosis-suspected patients, 51 were phenotypically and genotypically characterized as R. equi based on literature data. Mycolic acid analysis showed that all suspected R. equi had compounds with a retention factor (Rf between 0.4-0.5. Genotypic characterization indicated the presence of the choE gene 959 bp fragments in 51 isolates CAMP test positive. Twenty-two CAMP test negative isolates were negative for the choE gene. Five isolates presumptively identified as R. equi, CAMP test positive, were choE gene negative, and probably belonged to other bacterial species. CONCLUSIONS: The phenotypic and molecular techniques used constitute a good methodological tool to identify R. equi.

Full Text Available Rhodococcus equi (R. equi is an uncommon cause of infection in immunocompetent individuals. We describe a case of R. equi bacteremia associated with hemodialysis (HD catheter in an immunocompetent patient. A 38-year-old female with end-stage renal disease (ESRD of uncertain etiology, on HD for the past 15 months who was previously healthy otherwise, was admitted with the complaints of intermittent fever, mild nausea and occasional vomiting for two weeks. Last HD was performed four days earlier through a tunneled right internal jugular permacath. Clinically the patient was afebrile and in no acute distress. She was hemodynamically stable with no peripheral stigmata of an endovascular infection. Physical examination was essentially normal. Initially, the patient was treated with intravenous vancomycin with each HD, retaining the catheter. However, due to persistently positive blood cultures, HD catheter had to be removed. The patient became afebrile and nausea and vomiting resolved. She improved clinically, and repeated surveillance blood cultures done after the removal of catheter were reported negative. Subsequently, a new HD catheter was inserted for her. Although R. equi is an uncommon cause of infection in immunocompetent individuals, it does occur with considerable mortality and morbidity, and a high index of clinical suspicion is required to recognize this infection in immunocompetent individuals.

The aim was to determine the occurrence of Rhodococcus equi in equines and their environment in Jammu (R.S. Pura, Katra), molecular characterization and to determine the antibiotic resistance pattern of R. equi. A total of 96 nasopharyngeal swab samples were collected from equines. The organism was isolated on Columbia nalidixic acid agar containing 5% sheep blood as well as on sheep blood agar and was later confirmed by cultural characteristics and biochemical tests. Molecular detection of R. equi isolates was done by 16S rRNA gene amplification followed by virulence associated protein A (Vap A) gene amplification. Antibiogram was performed against five antibiotics, viz., amoxicillin, penicillin G, streptomycin, rifampicin, and methicillin. During the study, 9 R. equi isolates were identified on the basis of cultural and biochemical tests. In the polymerase chain reaction based detection, 3 among the 9 rhodococcal isolates were positive for species-specific 16S rRNA gene and revealed amplicon of 450 bp for confirmation of 16S rRNA gene. None of the sample was found positive for Vap A gene. In antibiogram, R. equi isolates were found sensitive for amoxicillin, while some isolates were also found resistant to the most conventional antibiotic penicillin G. From this study, it was concluded that R. equi infection is prevalent in equines in Jammu region of India and the indiscriminate use of the antibiotics is leading toward the development of resistant strains of R. equi.

Aim: The aim was to determine the occurrence of Rhodococcus equi in equines and their environment in Jammu (R.S. Pura, Katra), molecular characterization and to determine the antibiotic resistance pattern of R. equi. Materials and Methods: A total of 96 nasopharyngeal swab samples were collected from equines. The organism was isolated on Columbia nalidixic acid agar containing 5% sheep blood as well as on sheep blood agar and was later confirmed by cultural characteristics and biochemical tests. Molecular detection of R. equi isolates was done by 16S rRNA gene amplification followed by virulence associated protein A (Vap A) gene amplification. Antibiogram was performed against five antibiotics, viz., amoxicillin, penicillin G, streptomycin, rifampicin, and methicillin. Results: During the study, 9 R. equi isolates were identified on the basis of cultural and biochemical tests. In the polymerase chain reaction based detection, 3 among the 9 rhodococcal isolates were positive for species-specific 16S rRNA gene and revealed amplicon of 450 bp for confirmation of 16S rRNA gene. None of the sample was found positive for Vap A gene. In antibiogram, R. equi isolates were found sensitive for amoxicillin, while some isolates were also found resistant to the most conventional antibiotic penicillin G. Conclusion: From this study, it was concluded that R. equi infection is prevalent in equines in Jammu region of India and the indiscriminate use of the antibiotics is leading toward the development of resistant strains of R. equi. PMID:28246441

Presents a procedure by which cytokinins are used to induce a population of dividing and differentiating cells on the cut surface of the roots of an intact plant. Includes the method used, results, and suggestions for a variety of variables that may be tested. (RT)

Losses of one naturally occurring cytokinin (zeatin) and one synthetic cytoknin (kinetin) were determined during purification of turnips (Brassica compestris) infected by Plasmodiophora brassicae (clubroot). A known amount of zeatin and 8‐14C‐kinetin was added after homogenization of plant material

The catalytic reaction of cytokinin oxidase/dehydrogenase (EC 1.5.99.12) was studied in detail using the recombinant flavoenzyme from maize. Determination of the redox potential of the covalently linked flavin cofactor revealed a relatively high potential dictating the type of electron acceptor that

Losses of one naturally occurring cytokinin (zeatin) and one synthetic cytoknin (kinetin) were determined during purification of turnips (Brassica compestris) infected by Plasmodiophora brassicae (clubroot). A known amount of zeatin and 8‐14C‐kinetin was added after homogenization of plant material

Soils contaminated with o-xylene were more difficult to bioremediate than those contaminated with other BTEX hydrocarbons (benzene, toluene, ethylbenzene, m-xylene and p-xylene). In order to identify microorganisms responsible for o-xylene degradation in soil, microbial community structure analyses were carried out with two soil samples in the presence of o-xylene and mineral nutrients. In two different soil samples, Rhodococcus opacus became abundant. We were also able to isolate o-xylene degrading Rhodococcus species from these soil samples. A primer set was developed to specifically detect a cluster of this Rhodococcus group including isolated Rhodococcus strains, Rhodococcus opacus and Rhodococcus koreensis. The growth of this bacterial group in an o-xylene-contaminated soil was followed by competitive PCR (cPCR). The decrease in o-xylene clearly paralleled the growth of the Rhodococcus group.

The metabolism of cytokinins (CKs) and auxins in vascular plants is relatively well understood, but data concerning their metabolic pathways in non-vascular plants are still rather rare. With the aim of filling this gap, 20 representatives of taxonomically major lineages of cyanobacteria and algae from Cyanophyceae, Xanthophyceae, Eustigmatophyceae, Porphyridiophyceae, Chlorophyceae, Ulvophyceae, Trebouxiophyceae, Zygnematophyceae and Klebsormidiophyceae were analysed for endogenous profiles of CKs and auxins and some of them were used for studies of the metabolic fate of exogenously applied radiolabelled CK, [(3)H]trans-zeatin (transZ) and auxin ([(3)H]indole-3-acetic acid (IAA)), and the dynamics of endogenous CK and auxin pools during algal growth and cell division. Quantification of phytohormone levels was performed by high-performance or ultrahigh-performance liquid chromatography-electrospray tandem mass spectrometry (HPLC-MS/MS, UHPLC-MS/MS). The dynamics of exogenously applied [(3)H]transZ and [(3)H]IAA in cell cultures were monitored by HPLC with on-line radioactivity detection. The comprehensive screen of selected cyanobacteria and algae for endogenous CKs revealed a predominance of bioactive and phosphate CK forms while O- and N-glucosides evidently did not contribute greatly to the total CK pool. The abundance of cis-zeatin-type CKs and occurrence of CK 2-methylthio derivatives pointed to the tRNA pathway as a substantial source of CKs. The importance of the tRNA biosynthetic pathway was proved by the detection of tRNA-bound CKs during the course of Scenedesmus obliquus growth. Among auxins, free IAA and its oxidation catabolite 2-oxindole-3-acetic acid represented the prevailing endogenous forms. After treatment with [(3)H]IAA, IAA-aspartate and indole-3-acetyl-1-glucosyl ester were detected as major auxin metabolites. Moreover, different dynamics of endogenous CKs and auxin profiles during S. obliquus culture clearly demonstrated diverse roles of both

Full Text Available In this paper comparative genome and phenotype microarray analyses of Rhodococcus sp. BCP1 and Rhodococcus opacus R7 were performed. Rhodococcus sp. BCP1 was selected for its ability to grow on short-chain n-alkanes and R. opacus R7 was isolated for its ability to grow on naphthalene and on o-xylene. Results of genome comparison, including BCP1, R7, along with other Rhodococcus reference strains, showed that at least 30% of the genome of each strain presented unique sequences and only 50% of the predicted proteome was shared. To associate genomic features with metabolic capabilities of BCP1 and R7 strains, hundreds of different growth conditions were tested through Phenotype Microarray, by using Biolog plates and plates manually prepared with additional xenobiotic compounds. Around one-third of the surveyed carbon sources was utilized by both strains although R7 generally showed higher metabolic activity values compared to BCP1. Moreover, R7 showed broader range of nitrogen and sulphur sources. Phenotype Microarray data were combined with genomic analysis to genetically support the metabolic features of the two strains. The genome analysis allowed to identify some gene clusters involved in the metabolism of the main tested xenobiotic compounds. Results show that R7 contains multiple genes for the degradation of a large set of aromatic and PAHs compounds, while a lower variability in terms of genes predicted to be involved in aromatic degradation was found in BCP1. This genetic feature can be related to the strong genetic pressure exerted by the two different environment from which the two strains were isolated. According to this, in the BCP1 genome the smo gene cluster involved in the short-chain n-alkanes degradation, is included in one of the unique regions and it is not conserved in the Rhodococcus strains compared in this work. Data obtained underline the great potential of these two Rhodococcus spp. strains for biodegradation and

Modulating key dynamics of plant growth and development, the effects of the plant hormone cytokinin on animal cells gained much attention recently. Most previous studies on cytokinin effects on mammalian cells have been conducted with elevated cytokinin concentration (in the μM range). However, to examine physiologically relevant dose effects of cytokinins on animal cells, we systematically analyzed the impact of kinetin in cultured cells at low and high concentrations (1nM-10μM) and examined cytotoxic and genotoxic conditions. We furthermore measured the intrinsic antioxidant activity of kinetin in a cell-free system using the Ferric Reducing Antioxidant Power assay and in cells using the dihydroethidium staining method. Monitoring viability, we looked at kinetin effects in mammalian cells such as HL60 cells, HaCaT human keratinocyte cells, NRK rat epithelial kidney cells and human peripheral lymphocytes. Kinetin manifests no antioxidant activity in the cell free system and high doses of kinetin (500 nM and higher) reduce cell viability and mediate DNA damage in vitro. In contrast, low doses (concentrations up to 100 nM) of kinetin confer protection in cells against oxidative stress. Moreover, our results show that pretreatment of the cells with kinetin significantly reduces 4-nitroquinoline 1-oxide mediated reactive oxygen species production. Also, pretreatment with kinetin retains cellular GSH levels when they are also treated with the GSH-depleting agent patulin. Our results explicitly show that low kinetin doses reduce apoptosis and protect cells from oxidative stress mediated cell death. Future studies on the interaction between cytokinins and human cellular pathway targets will be intriguing. PMID:28005918

Full Text Available Plant responses to phytohormone stimuli are the most important biological features for plants to survive in a complex environment. Cytokinin regulates growth and nutrient homeostasis, such as the phosphate (Pi starvation response and Pi uptake in plants. However, the mechanisms underlying how cytokinin participates in Pi uptake and Pi signaling are largely unknown. In this study, we found that OsARF16 is required for the cytokinin response and is involved in the negative regulation of Pi uptake and Pi signaling by cytokinin.The mutant osarf16 showed an obvious resistance to exogenous cytokinin treatment and the expression level of the OsARF16 gene was considerably up-regulated by cytokinin. Cytokinin (6-BA application suppressed Pi uptake and the Pi starvation response in wild-type Nipponbare (NIP and all these responses were compromised in the osarf16 mutant. Our data showed that cytokinin inhibits the transport of Pi from the roots to the shoots and that OsARF16 is involved in this process. The Pi content in the osarf16 mutant was much higher than in NIP under 6-BA treatment. The expressions of PHOSPHATE TRANSPORTER1 (PHT1 genes, phosphate (Pi starvation-induced (PSI genes and purple PAPase genes were higher in the osarf16 mutant than in NIP under cytokinin treatment.Our results revealed a new biological function for OsARF16 in the cytokinin-mediated inhibition of Pi uptake and Pi signaling in rice.

When grown in the absence of light, the det1 mutants of Arabidopsis thaliana develop characteristics of light-grown plants by morphological, cellular, and molecular criteria. Further, in light-grown plants, mutations in the DET1 gene affect cell-type-specific expression of light-regulated genes and the chloroplast developmental program. Here we show that the addition of exogenously added cytokinins (either 2-isopentenyl adenine, kinetin, or benzyladenine) to the growth medium of dark-germinated wild-type seedlings results in seedlings that resemble det1 mutants, instead of having the normal etiolated morphology. Like det1 mutants, these dark-grown seedlings now contain chloroplasts and have high levels of expression of genes that are normally ``light``-regulated. These results suggest an important role for cytokinins during greening of Arabidopsis, and may implicate cytokinin levels or an increased sensitivity to cytokinins as explanations for some of the observed phenotypes of det1 mutants.

The phytohormones, abscisic acid and cytokinin, once were thought to be present uniquely in plants, but increasing evidence suggests that these hormones are present in a wide variety of organisms. Few studies have examined fungi for the presence of these "plant" hormones or addressed whether their levels differ based on the nutrition mode of the fungus. This study examined 20 temperate forest fungi of differing nutritional modes (ectomycorrhizal, wood-rotting, saprotrophic). Abscisic acid and cytokinin were present in all fungi sampled; this indicated that the sampled fungi have the capacity to synthesize these two classes of phytohormones. Of the 27 cytokinins analyzed by HPLC-ESI MS/MS, seven were present in all fungi sampled. This suggested the existence of a common cytokinin metabolic pathway in fungi that does not vary among different nutritional modes. Predictions regarding the source of isopentenyl, cis-zeatin and methylthiol CK production stemming from the tRNA degradation pathway among fungi are discussed.

Rhodococcus (R) equi is an opportunistic, uncommon human pathogen that causes mainly infection in immunocompromised hosts. The disease is usually presented as subacute pneumonia that is mostly cavitary and sometimes bacteremic. We reported the extremly rare case of a 43-year-old woman with Hodgkin lymphoma, who developed R equi pulmonary infection after recieving multiple courses of chemotherapy. Secondary, the patient developed bacteremia, leading to sepsis and dissemination of R equi infection in many extrapulmonary sites. At addmission the patient was febrile, tachypnoic, tachycardic, hypotensive, with fa cial edema, splenomegaly, positive meningeal signs, left hemiparesis and paraparesis. Laboratory data included erythrocyte sedimentation rate (ESR) > 140 mm/h, C-reactive protein (CRP) 143.0 mg/L, red blood cells (RBC) 2.14 x 10(12)/L, whyite blood cells (WBC) 2.8 x 10(9)/L, lactate dehydrogenase (LDH) 706 U/L, serum albumin 26 g/L, sodium 127 mmol/L and potassium 2.7 mmol/L. Blood culture and culture of sputum and empyema were positive for R equi. Imaging studies demonstrated a large right cavitary pneumonia and abscess, empyema, pericarditis, mediastinal and intra-abdominal lymphadenopathy, brain and psoas abscesses, osteomyelitis and spondylodiscitis. The patient recovered completely after a 12-month treatment with combinations of parenteral and oral antibiotics (meropenem, vancomycin, teicoplanin, ciprofloxacin, rifampicin, macrolides etc), including drainage of abscesses and empyema. Eight years after completition of the treatment the patient was without recurrence of R equi infection and lymphoma. Since the eradication od R equi is very difficult, it is very important to make the diagnosis and initiate appropriate antibiotic therapy as soon as possible.

Sedentary plant-parasitic cyst nematodes are biotrophs that cause significant losses in agriculture. Parasitism is based on modifications of host root cells that lead to the formation of a hypermetabolic feeding site (a syncytium) from which nematodes withdraw nutrients. The host cell cycle is activated in an initial cell selected by the nematode for feeding, followed by activation of neighboring cells and subsequent expansion of feeding site through fusion of hundreds of cells. It is generally assumed that nematodes manipulate production and signaling of the plant hormone cytokinin to activate cell division. In fact, nematodes have been shown to produce cytokinin in vitro; however, whether the hormone is secreted into host plants and plays a role in parasitism remained unknown. Here, we analyzed the spatiotemporal activation of cytokinin signaling during interaction between the cyst nematode, Heterodera schachtii, and Arabidopsis using cytokinin-responsive promoter:reporter lines. Our results showed that cytokinin signaling is activated not only in the syncytium but also in neighboring cells to be incorporated into syncytium. An analysis of nematode infection on mutants that are deficient in cytokinin or cytokinin signaling revealed a significant decrease in susceptibility of these plants to nematodes. Further, we identified a cytokinin-synthesizing isopentenyltransferase gene in H. schachtii and show that silencing of this gene in nematodes leads to a significant decrease in virulence due to a reduced expansion of feeding sites. Our findings demonstrate the ability of a plant-parasitic nematode to synthesize a functional plant hormone to manipulate the host system and establish a long-term parasitic interaction.

Sedentary plant-parasitic cyst nematodes are biotrophs that cause significant losses in agriculture. Parasitism is based on modifications of host root cells that lead to the formation of a hypermetabolic feeding site (a syncytium) from which nematodes withdraw nutrients. The host cell cycle is activated in an initial cell selected by the nematode for feeding, followed by activation of neighboring cells and subsequent expansion of feeding site through fusion of hundreds of cells. It is generally assumed that nematodes manipulate production and signaling of the plant hormone cytokinin to activate cell division. In fact, nematodes have been shown to produce cytokinin in vitro; however, whether the hormone is secreted into host plants and plays a role in parasitism remained unknown. Here, we analyzed the spatiotemporal activation of cytokinin signaling during interaction between the cyst nematode, Heterodera schachtii, and Arabidopsis using cytokinin-responsive promoter:reporter lines. Our results showed that cytokinin signaling is activated not only in the syncytium but also in neighboring cells to be incorporated into syncytium. An analysis of nematode infection on mutants that are deficient in cytokinin or cytokinin signaling revealed a significant decrease in susceptibility of these plants to nematodes. Further, we identified a cytokinin-synthesizing isopentenyltransferase gene in H. schachtii and show that silencing of this gene in nematodes leads to a significant decrease in virulence due to a reduced expansion of feeding sites. Our findings demonstrate the ability of a plant-parasitic nematode to synthesize a functional plant hormone to manipulate the host system and establish a long-term parasitic interaction. PMID:26417108

For decades the burning of fossil fuels released a lot of pollutants in the atmosphere. Among the most harmful is sulfur dioxide (SO{sub 2}), which reacts with the moisture in the air and turns into sulfuric acid, being the main cause of acid rain. Acid rain is very harmful to animal and plant kingdoms; accelerates the corrosion's processes of buildings and monuments, and causes serious health problems for humans. As a result, many countries have reformed their legislation to require the sale of fuels with very low sulfur content. The existing processes of desulfurization are not capable of removing sulfur so low. Therefore, there has developed a new process called bio desulfurization. In this process, the degradation of sulfur occurs through the action of microorganisms that act as catalysts. The bacterium Rhodococcus erythropolis has emerged as one of the most promising for bio desulfurization because it removes the sulfur without breaking the benzene rings, thereby maintaining the potential energy of the same. Using dibenzothiophene as a model of sulfur compounds, the products of the bio desulfurization process are 2- hydroxybiphenyl and sulfate. In this study we sought to examine the desulfurizing capacity of national Rhodococcus erythropolis strain ATCC4277 in a batch reactor using concentrations of organic phase (n-dodecane) of 20 and 80% (v/v). Rhodococcus erythropolis ATCC4277 was capable of degrading DBT in 93.3 and 98.0% in the presence of 20 and 80% (v/v) of synthetic fuel, respectively. (author)

We conducted a case–control study of 18 US transplant recipients with Rhodococcus infection and 36 matched controls. The predominant types of infection were pneumonia and bacteremia. Diabetes mellitus and recent opportunistic infection were independently associated with disease. Outcomes were generally favorable except for 1 relapse and 1 death. PMID:28221102

Rhodococcus equi, mainly known from veterinary medicine as a pathogen in domestic animals, can also cause infections in immunocompromised humans, especially in those with defects in cellular immunity. Alemtuzumab, an anti-CD52 monoclonal antibody, causes lymphocytopenia by eliminating CD52-positive

We report a draft genome sequence of Rhodococcus ruber IEGM 231, isolated from a water spring near an oil-extracting enterprise (Perm region, Russian Federation). This sequence provides important insights into the genetic mechanisms of propane and n-butane metabolism, organic sulfide and beta-sitosterol biotransformation, glycolipid biosurfactant production, and heavy metal resistance in actinobacteria.

We report a draft genome sequence of Rhodococcus ruber IEGM 231, isolated from a water spring near an oil-extracting enterprise (Perm region, Russian Federation). This sequence provides important insights into the genetic mechanisms of propane and n-butane metabolism, organic sulfide and beta-sitosterol biotransformation, glycolipid biosurfactant production, and heavy metal resistance in actinobacteria.

Rhodococcus equi is increasingly recognized as an opportunistic pathogen in solid organ transplant recipients. Primary pulmonary involvement is the most common finding. We report a case of a 42-year-old female kidney transplant recipient who developed multiple disseminated abscesses caused by R. equ

The genes involved in isoprene (2-methyl-1,3-butadiene) utilization in Rhodococcus sp. strain AD45 were cloned and characterized. Sequence analysis of an 8.5-kb DNA fragment showed the presence of 10 genes of which 2 encoded enzymes which were previously found to be involved in isoprene degradation:

A gene encoding a putrescine oxidase (PuORh, EC 1.4.3.10) was identified from the genome of Rhodococcus erythropolis NCIMB 11540. The gene was cloned in the pBAD vector and overexpressed at high levels in Escherichia coli. The purified enzyme was shown to be a soluble dimeric flavoprotein consisting

A gene encoding a putrescine oxidase (PuORh, EC 1.4.3.10) was identified from the genome of Rhodococcus erythropolis NCIMB 11540. The gene was cloned in the pBAD vector and overexpressed at high levels in Escherichia coli. The purified enzyme was shown to be a soluble dimeric flavoprotein consisting

Rhodococcus equi is increasingly recognized as an opportunistic pathogen in solid organ transplant recipients. Primary pulmonary involvement is the most common finding. We report a case of a 42-year-old female kidney transplant recipient who developed multiple disseminated abscesses caused by R.

Rhodococcus (R.) equi is a facultative intracellular pathogen that causes severe pyogranulomatous pneumonia in foals up to 5 months of age. Despite the great need for a prophylactic measure against this devastating disease, no commercial vaccine is available. Today only long-term and cumbersome

Rhodococcus equi, mainly known from veterinary medicine as a pathogen in domestic animals, can also cause infections in immunocompromised humans, especially in those with defects in cellular immunity. Alemtuzumab, an anti-CD52 monoclonal antibody, causes lymphocytopenia by eliminating CD52-positive

Full Text Available Cholesterol oxidase (CHO is one of the valuable enzymes that play an important role in: measurement of serum cholesterol, food industry as a biocatalyst and agriculture as a biological larvicide. This enzyme was produced by several bacterial strains. Wild type enzyme produced by Rhodococcus sp. secret two forms of CHO enzyme: extra cellular and membrane bound type which its amount is low and unstable. The goal of the study was cloning, expression, and enzymatic activity evaluation of cholesterol oxidase gene isolated from a native Rhodococcus sp. CHO gene was isolated from native bacteria and cloned into pET23a. In the next step, the construct was expressed in E.coli BL21 and induced by different concentration of IPTG ranges from 0.1 - 0.9 mM. This gene contains 1642 bp and encodes a protein consists of 533 amino acids. It has about 96 % homology with CHO gene isolated from Rhodococcus equi. The high expression was obtained in 0.5 mM concentration of IPTG after 4 hour induction. This recombinant enzyme had a molecular weight of 55 kDa, that secretion of intra cellular type is much more than extracellular form. The optimum pH and temperature conditions for the recombinant enzyme were 7.5 and 45°C, respectively. CHO enzyme obtained from Rhodococcus sp. is a cheap enzyme with medical and industrial applications that can be produced easily and purified in large scale with simple methods.

The genome of Rhodococcus jostii RHA1 contains an unusually large number of oxygenase encoding genes. Many of these genes have yet an unknown function, implying that a notable part of the biochemical and catabolic biodiversity of this Gram-positive soil actinomycete is still elusive. Here we present

Background: Bacteria of the genus Rhodococcus are well known for their ability to degrade a large range of organic compounds. Some rhodococci are free-living, saprophytic bacteria; others are animal and plant pathogens. Recently, several studies have shown that their genomes encode putative pathways

BACKGROUND: Bacteria of the genus Rhodococcus are well known for their ability to degrade a large range of organic compounds. Some rhodococci are free-living, saprophytic bacteria; others are animal and plant pathogens. Recently, several studies have shown that their genomes encode putative pathways

Full Text Available In Arabidopsis thaliana, lateral roots (LRs initiate from anticlinal cell divisions of pericycle founder cells. The formation of LR primordia is regulated antagonistically by the phytohormones cytokinin and auxin. It has previously been shown that cytokinin has an inhibitory effect on the patterning events occurring during LR formation. However, the molecular players involved in cytokinin repression are still unknown. In a similar manner to protoxylem formation in Arabidopsis roots, in which AHP6 (ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEIN 6 acts as a cytokinin inhibitor, we reveal that AHP6 also functions as a cytokinin repressor during early stages of LR development. We show that AHP6 is expressed at different developmental stages during LR formation and is required for the correct orientation of cell divisions at the onset of LR development. Moreover, we demonstrate that AHP6 influences the localization of the auxin efflux carrier PIN1, which is necessary for patterning the LR primordia. In summary, we show that the inhibition of cytokinin signaling through AHP6 is required to establish the correct pattern during LR initiation.

Full Text Available Auxin and cytokinin signaling participates in regulating a large spectrum of developmental and physiological processes in plants. The shoots and roots of plants have specific and sometimes even contrary responses to these hormones. Recent studies have clearly shown that establishing the spatiotemporal distribution of auxin and cytokinin response signals is central for the control of shoot apical meristem (SAM induction in cultured tissues. However, little is known about the role of these hormones in root apical meristem (RAM initiation. Here, we found that the expression patterns of several regulatory genes critical for RAM formation were correlated with the establishment of the embryonic root meristem during somatic embryogenesis in Arabidopsis. Interestingly, the early expression of the WUS-RELATED HOMEOBOX 5 (WOX5 and WUSCHEL (WUS genes was induced and was nearly overlapped within the embryonic callus when somatic embryos (SEs could not be identified morphologically. Their correct expression was essential for RAM and SAM initiation and embryonic shoot–root axis establishment. Furthermore, we analyzed the auxin and cytokinin response during SE initiation. Notably, cytokinin response signals were detected in specific regions that were correlated with induced WOX5 expression and subsequent SE formation. Overexpression of the ARABIDOPSIS RESPONSE REGULATOR genes ARR7 and ARR15 (feedback repressors of cytokinin signaling, disturbed RAM initiation and SE induction. These results provide new information on auxin and cytokinin-regulated apical–basal polarity formation of shoot–root axis during somatic embryogenesis.

Cytokinins (CKs) regulate plant growth and development via a complex network of CK signaling. Here, we perform functional analyses with CK-deficient plants to provide direct evidence that CKs negatively regulate salt and drought stress signaling. All CK-deficient plants with reduced levels of various CKs exhibited a strong stress-tolerant phenotype that was associated with increased cell membrane integrity and abscisic acid (ABA) hypersensitivity rather than stomatal density and ABA-mediated stomatal closure. Expression of the Arabidopsis thaliana ISOPENTENYL-TRANSFERASE genes involved in the biosynthesis of bioactive CKs and the majority of the Arabidopsis CYTOKININ OXIDASES/DEHYDROGENASES genes was repressed by stress and ABA treatments, leading to a decrease in biologically active CK contents. These results demonstrate a novel mechanism for survival under abiotic stress conditions via the homeostatic regulation of steady state CK levels. Additionally, under normal conditions, although CK deficiency increased the sensitivity of plants to exogenous ABA, it caused a downregulation of key ABA biosynthetic genes, leading to a significant reduction in endogenous ABA levels in CK-deficient plants relative to the wild type. Taken together, this study provides direct evidence that mutual regulation mechanisms exist between the CK and ABA metabolism and signals underlying different processes regulating plant adaptation to stressors as well as plant growth and development. PMID:21719693

Groundwater contamination by the explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is a global problem. Israel's coastal aquifer was contaminated with RDX. This aquifer is mostly aerobic and we therefore sought aerobic bacteria that might be involved in natural attenuation of the compound in the aquifer. RDX-degrading bacteria were captured by passively sampling the indigenous bacteria onto sterile sediments placed within sampling boreholes. Aerobic RDX biodegradation potential was detected in the sediments sampled from different locations along the plume. RDX degradation with the native sampled consortium was accompanied by 4-nitro-2,4-diazabutanal formation. Two bacterial strains of the genus Rhodococcus were isolated from the sediments and identified as aerobic RDX degraders. The xplA gene encoding the cytochrome P450 enzyme was partially (~500 bp) sequenced from both isolates. The obtained DNA sequences had 99% identity with corresponding gene fragments of previously isolated RDX-degrading Rhodococcus strains. RDX degradation by both strains was prevented by 200 μM of the cytochrome P450 inhibitor metyrapone, suggesting that cytochrome P450 indeed mediates the initial step in RDX degradation. RDX biodegradation activity by the T7 isolate was inhibited in the presence of nitrate or ammonium concentrations above 1.6 and 5.5 mM, respectively (100 mg l(-1)) while the T9N isolate's activity was retarded only by ammonium concentrations above 5.5 mM. This study shows that bacteria from the genus Rhodococcus, potentially degrade RDX in the saturated zone as well, following the same aerobic degradation pathway defined for other Rhodococcus species. RDX-degrading activity by the Rhodococcus species isolate T9N may have important implications for the bioremediation of nitrate-rich RDX-contaminated aquifers.

A cell culture of anise was grown in the presence or absence of 2,4-dichlorophenoxyacetic acid (2,4-D). Application of isopentenyladenine or isopentenyladenosine (4·10(-8) to 4·10(-7) M) to the proembryonic culture (+2,4-D) yielded an increase of the cell density, in contrast to a proembryonic culture grown without exogenous application of cytokinins. Embryogenesis was induced by transferring the cells to a hormone-free medium. Embryo development was promoted by isopentenyladenine and isopentenyladenosine (5·10(-8) to 5·10(-7) M), higher concentrations (5·10(-6) M) inhibited embryogenesis. The effect of cytokinins on embryogenesis was only promotive until the third day of culture, i.e. coincident with cell growth rather than differentiation.

immunity has been described in Arabidopsis, rice, and tobacco. Although interactions of cytokinins with salicylic acid and auxin have been indicated, the complete network of cytokinin interactions with other immunity-relevant phytohormones is not yet understood. Therefore, we studied the interaction...... of kinetin and abscisic acid as a negative regulator of plant immunity to modulate resistance in tobacco against Pseudomonas syringae. By analyzing infection symptoms, pathogen proliferation, and accumulation of the phytoalexin scopoletin as a key mediator of kinetin-induced resistance in tobacco......, antagonistic interaction of these phytohormones in plant immunity was identified. Kinetin reduced abscisic acid levels in tobacco, while increased abscisic acid levels by exogenous application or inhibition of abscisic acid catabolism by diniconazole neutralized kinetin-induced resistance. Based...

Rhodococcus equi causes fatal pyogranulomatous pneumonia in foals and immunocompromised animals and humans. Despite its importance, there is currently no effective vaccine against the disease. The actinobacteria R. equi and the human pathogen Mycobacterium tuberculosis are related, and both cause

Two-component flavoprotein monooxygenases are emerging biocatalysts that generally consist of a monooxygenase and a reductase component. Here we show that Rhodococcus opacus 1CP encodes a multifunctional enantioselective flavoprotein monooxygenase system composed of a single styrene monooxygenase (S

Here, we present the complete genome of Rhodococcus pyridinivorans SB3094, a methyl-ethyl-ketone (MEK)-degrading strain used for bioaugmentation relating to the treatment of wastewater contamination with petrochemical hydrocarbons. The genome highlights important features for bioaugmentation...

Auxin produced by an active primary shoot apex is transported down the main stem and inhibits the growth of the axillary buds below it, contributing to apical dominance. Here we use Arabidopsis thaliana cytokinin (CK) biosynthetic and signalling mutants to probe the role of CK in this process. It is well established that bud outgrowth is promoted by CK, and that CK synthesis is inhibited by auxin, leading to the hypothesis that release from apical dominance relies on an increased supply of CK to buds. Our data confirm that decapitation induces the expression of at least one ISOPENTENYLTRANSFERASE (IPT) CK biosynthetic gene in the stem. We further show that transcript abundance of a clade of the CK-responsive type-A Arabidopsis response regulator (ARR) genes increases in buds following CK supply, and that, contrary to their typical action as inhibitors of CK signalling, these genes are required for CK-mediated bud activation. However, analysis of the relevant arr and ipt multiple mutants demonstrates that defects in bud CK response do not affect auxin-mediated bud inhibition, and increased IPT transcript levels are not needed for bud release following decapitation. Instead, our data suggest that CK acts to overcome auxin-mediated bud inhibition, allowing buds to escape apical dominance under favourable conditions, such as high nitrate availability. Significance Statement It has been proposed that the release of buds from auxin-mediated apical dominance following decapitation requires increased cytokinin biosynthesis and consequent increases in cytokinin supply to buds. Here we show that in Arabidopsis, increases in cytokinin appear to be unnecessary for the release of buds from apical dominance, but rather allow buds to escape the inhibitory effect of apical auxin, thereby promoting bud activation in favourable growth conditions. PMID:25904120

We studied the role of Arabidopsis thaliana TCP15, a member of the TEOSINTE BRANCHED1-CYCLOIDEA-PCF (TCP) transcription factor family, in gynoecium development. Plants that express TCP15 from the 35S CaMV promoter (35S:TCP15) develop flowers with defects in carpel fusion and a reduced number of stigmatic papillae. In contrast, the expression of TCP15 fused to a repressor domain from its own promoter causes the development of outgrowths topped with stigmatic papillae from the replum. 35S:TCP15 plants show lower levels of the auxin indoleacetic acid and reduced expression of the auxin reporter DR5 and the auxin biosynthesis genes YUCCA1 and YUCCA4, suggesting that TCP15 is a repressor of auxin biosynthesis. Treatment of plants with cytokinin enhances the developmental effects of expressing TCP15 or its repressor form. In addition, treatment of a knock-out double mutant in TCP15 and the related gene TCP14 with cytokinin causes replum enlargement, increased development of outgrowths, and the induction of the auxin biosynthesis genes YUCCA1 and YUCCA4. A comparison of the phenotypes observed after cytokinin treatment of plants with altered expression levels of TCP15 and auxin biosynthesis genes suggests that TCP15 modulates gynoecium development by influencing auxin homeostasis. We propose that the correct development of the different tissues of the gynoecium requires a balance between auxin levels and cytokinin responses, and that TCP15 participates in a feedback loop that helps to adjust this balance.

Full Text Available It was found that the hardwood form of Cantharellus cibarius (strain 5400 produced less cytokinin-like substances than the coniferous form (strain 5410. Among the active substances the following were detected: 2iP, 2iP riboside and zeatin. No significant differences in ethylene production between both strains in the presence or absence of methionine (considered to be the precursor of this gas were noted.

Rhodococcus sp. strain DK17 was isolated from soil and analyzed for the ability to grow on o-xylene as the sole carbon and energy source. Although DK17 cannot grow on m- and p-xylene, it is capable of growth on benzene, phenol, toluene, ethylbenzene, isopropylbenzene, and other alkylbenzene isomers. One UV-generated mutant strain, DK176, simultaneously lost the ability to grow on o-xylene, ethylbenzene, isopropylbenzene, toluene, and benzene, although it could still grow on phenol. The mutant strain was also unable to oxidize indole to indigo following growth in the presence of o-xylene. This observation suggests the loss of an oxygenase that is involved in the initial oxidation of the (alkyl)benzenes tested. Another mutant strain, DK180, isolated for the inability to grow on o-xylene, retained the ability to grow on benzene but was unable to grow on alkylbenzenes due to loss of a meta-cleavage dioxygenase needed for metabolism of methyl-substituted catechols. Further experiments showed that DK180 as well as the wild-type strain DK17 have an ortho-cleavage pathway which is specifically induced by benzene but not by o-xylene. These results indicate that DK17 possesses two different ring-cleavage pathways for the degradation of aromatic compounds, although the initial oxidation reactions may be catalyzed by a common oxygenase. Gas chromatography-mass spectrometry and 300-MHz proton nuclear magnetic resonance spectrometry clearly show that DK180 accumulates 3,4-dimethylcatechol from o-xylene and both 3- and 4-methylcatechol from toluene. This means that there are two initial routes of oxidation of toluene by the strain. Pulsed-field gel electrophoresis analysis demonstrated the presence of two large megaplasmids in the wild-type strain DK17, one of which (pDK2) was lost in the mutant strain DK176. Since several other independently derived mutant strains unable to grow on alkylbenzenes are also missing pDK2, the genes encoding the initial steps in alkylbenzene

Arabidopsis has three cytokinin receptors genes: CRE1, AHK2, and AHK3. Availability of plants that are homozygous mutant for these three genes indicates that cytokinin receptors in the haploid cells are dispensable for the development of male and female gametophytes. The triple mutants form a few flowers but never set seed, indicating that reproductive growth is impaired. We investigated which reproductive processes are affected in the triple mutants. Anthers of mutant plants contained fewer pollen grains and did not dehisce. Pollen in the anthers completed the formation of the one vegetative nucleus and the two sperm nuclei, as seen in wild type. The majority of the ovules were abnormal: 78% lacked the embryo sac, 10% carried a female gametophyte that terminated its development before completing three rounds of nuclear division, and about 12% completed three rounds of nuclear division but the gametophytes were smaller than those of the wild type. Reciprocal crosses between the wild type and the triple mutants indicated that pollen from mutant plants did not germinate on wild-type stigmas, and wild-type pollen did not germinate on mutant stigmas. These results suggest that cytokinin receptors in the sporophyte are indispensable for anther dehiscence, pollen maturation, induction of pollen germination by the stigma, and female gametophyte formation and maturation.

The development of seeds in flowering plants is placed under complex interactions between maternal tissues, the embryo, and the endosperm. The endosperm plays a major role in the regulation of seed size. In Arabidopsis thaliana, endosperm size depends on the coordination of the genetic pathway HAIKU (IKU) with epigenetic controls comprising genome dosage, DNA methylation, and trimethylated lysine 27 on histone H3 (H3K27me3) deposition. However, the effectors that integrate these pathways have remained unknown. Here, we identify a target of the IKU pathway, the cytokinin oxidase CKX2, that affects cytokinin signaling. CKX2 expression is activated by the IKU transcription factor WRKY10 directly and promotes endosperm growth. CKX2 expression also depends on H3K27me3 deposition, which fluctuates in response to maternal genome dosage imbalance and DNA demethylation of male gametes. Hence, the control of endosperm growth by CKX2 integrates genetic and epigenetic regulations. In angiosperms, cytokinins are highly active in endosperm, and we propose that IKU effectors coordinate environmental and physiological factors, resulting in modulation of seed size.

Full Text Available The effect of two cytokinins (BAP, m-Topolin tested singly or in combination with a mixture of amino acids (glycine, glutamine, cysteine on multiplication rate and quality of Pelargonium x hederaefolium and Pelargonium x hortorum shoots were investigated. For both Pelargonium cultivars, the application of cytokinins greatly increased the production of axillary and adventitious shoots compared with the shoots produced on the medium without growth regulators. In the case of P. x hederaefolium no significant differences it were found in the number of shoot formed for BAP and m-Topolin treatments. For P. x hortorum the most effective multiplication of shoots was obtained on the medium supplemented with m-Topolin (1.0 mg l-1. Moreover, in the presence of m-Topolin an improvement of shoot quality of both genotypes has been observed (well developed shoots with soft green leaves. It was particularly apparent for P. x hortorum shoots. The addition of amino acids to a cytokinin medium increased slightly the number of formed shoots of P. x hederaefolium and influenced the shoot quality of both Pelargonium cultivars (well developed leaf blades, uniform growth of shoots in clumps, slower process of shoot senescence.

We investigated the effects of P(SARK)IPT (for Senescence-Associated Receptor KinaseIsopentenyltransferase) expression and cytokinin production on several aspects of photosynthesis in transgenic tobacco (Nicotiana tabacum cv SR1) plants grown under optimal or restricted (30% of optimal) watering regimes. There were no significant differences in stomatal conductance between leaves from wild-type and transgenic P(SARK)-IPT plants grown under optimal or restricted watering. On the other hand, there was a significant reduction in the maximum rate of electron transport as well as the use of triose-phosphates only in wild-type plants during growth under restricted watering, indicating a biochemical control of photosynthesis during growth under water deficit. During water deficit conditions, the transgenic plants displayed an increase in catalase inside peroxisomes, maintained a physical association among chloroplasts, peroxisomes, and mitochondria, and increased the CO(2) compensation point, indicating the cytokinin-mediated occurrence of photorespiration in the transgenic plants. The contribution of photorespiration to the tolerance of transgenic plants to water deficit was also supported by the increase in transcripts coding for enzymes involved in the conversion of glycolate to ribulose-1,5-bisphosphate. Moreover, the increase in transcripts indicated a cytokinin-induced elevation in photorespiration, suggesting the contribution of photorespiration in the protection of photosynthetic processes and its beneficial role during water stress.

Legume nodules result from coordinated interactions between the plant and nitrogen-fixing rhizobia. The phytohormone cytokinin promotes nodule formation, and recent findings suggest that the phytohormone auxin inhibits nodule formation. Here we show that microRNA160 (miR160) is a key signaling element that determines the auxin/cytokinin balance during nodule development in soybean (Glycine max). miR160 appears to promote auxin activity by suppressing the levels of the ARF10/16/17 family of repressor ARF transcription factors. Using quantitative PCR assays and a fluorescence miRNA sensor, we show that miR160 levels are relatively low early during nodule formation and high in mature nodules. We had previously shown that ectopic expression of miR160 in soybean roots led to a severe reduction in nodule formation, coupled with enhanced sensitivity to auxin and reduced sensitivity to cytokinin. Here we show that exogenous cytokinin restores nodule formation in miR160 over-expressing roots. Therefore, low miR160 levels early during nodule development favor cytokinin activity required for nodule formation. Suppression of miR160 levels using a short tandem target mimic (STTM160) resulted in reduced sensitivity to auxin and enhanced sensitivity to cytokinin. In contrast to miR160 over-expressing roots, STTM160 roots had increased nodule formation, but nodule maturation was significantly delayed. Exogenous auxin partially restored proper nodule formation and maturation in STTM160 roots, suggesting that high miR160 activity later during nodule development favors auxin activity and promotes nodule maturation. Therefore, miR160 dictates developmental stage-specific sensitivities to auxin and cytokinin to direct proper nodule formation and maturation in soybean.

Full Text Available Abstract Background Riboswitches are mRNA elements that change conformation when bound to small molecules. They are known to be key regulators of biosynthetic pathways in both prokaryotes and eukaryotes. Presentation of the Hypothesis The hypothesis presented here is that riboswitches function as receptors in hormone perception. We propose that riboswitches initiate or integrate signaling cascades upon binding to classic signaling molecules. The molecular interactions for ligand binding and gene expression control would be the same as for biosynthetic pathways, but the context and the cadre of ligands to consider is dramatically different. The hypothesis arose from the observation that a compound used to identify adenine binding RNA sequences is chemically similar to the classic plant hormone, or growth regulator, cytokinin. A general tenet of the hypothesis is that riboswitch-binding metabolites can be used to make predictions about chemically related signaling molecules. In fact, all cell permeable signaling compounds can be considered as potential riboswitch ligands. The hypothesis is plausible, as demonstrated by a cursory review of the transcriptome and genome of the model plant Arabidopsis thaliana for transcripts that i contain an adenine aptamer motif, and ii are also predicted to be cytokinin-regulated. Here, one gene, CRK10 (for Cysteine-rich Receptor-like Kinase 10, At4g23180, contains an adenine aptamer-related sequence and is down-regulated by cytokinin approximately three-fold in public gene expression data. To illustrate the hypothesis, implications of cytokinin-binding to the CRK10 mRNA are discussed. Testing the hypothesis At the broadest level, screening various cell permeable signaling molecules against random RNA libraries and comparing hits to sequence and gene expression data bases could determine how broadly the hypothesis applies. Specific cases, such as CRK10 presented here, will require experimental validation of direct

Cytokinins are phytohormones that play key roles in the maintenance of stem cell activity in plants. Although alternative single-step and two-step activation pathways for cytokinin have been proposed, the significance of the single-step pathway which is catalyzed by LONELY GUY (LOG), is not fully understood. We analyzed the metabolic flow of cytokinin activation in Arabidopsis log multiple mutants using stable isotope-labeled tracers and characterized the mutants' morphological and developmental phenotypes. In tracer experiments, cytokinin activation was inhibited most pronouncedly by log7, while the other log mutations had cumulative effects. Although sextuple or lower-order mutants did not show drastic phenotypes in vegetative growth, the log1log2log3log4log5log7log8 septuple T-DNA insertion mutant in which the LOG-dependent pathway is impaired, displayed severe retardation of shoot and root growth with defects in the maintenance of the apical meristems. Detailed observation of the mutants showed that LOG7 was required for the maintenance of shoot apical meristem size. LOG7 was also suggested to play a role for normal primary root growth together with LOG3 and LOG4. These results suggest a dominant role of the single-step activation pathway mediated by LOGs for cytokinin production, and overlapping but differentiated functions of the members of the LOG gene family in growth and development.

The gene for new Rhodococcus erythropolis TA37 acylamidase, which possesses unique substrate specificity, has been cloned and expressed in E. coli. Substrates for this enzyme are not only simple amides, such as acetamide and propionamide, but also N-substituted amides, such as 4'-nitroacetanilide. The 1431-bp gene was expressed in E. coli BL21 (DE3) cells on pET16b plasmid under the control of a promoter of the φ 10 gene from the T7 phage. The molecular mass of recombinant acylamidase in E. coli was 55 kDa, which corresponded to that of native acylamidase from Rhodococcus erythropolis TA37. Recombinant acylamidase was able to hydrolize N-substituted amides. A search of a nucleotide database and multiple alignment revealed that acylamidase belonged to the Amidase protein family PF01425, but its nucleotide and amino acid sequences differed significantly from those of the described amidases.

Rhodococcus equi, previously called Corynebacterium equi, is known to cause pneumonia in foals and swine. Although it was known to cause infection rarely in humans, R equi infection in humans has increased with the advent of HIV and increased use of immunosuppressants. We report a case of a 48-year-old male patient with newly diagnosed HIV/AIDS presenting with recurrent R equi bacteremia, pericardial effusion, and pericardial cyst. The infection was treated with drainage of the pericardial effusion and cyst and 2 weeks of intravenous vancomycin and 6 months of oral azithromycin and levofloxacin. Rhodococcus equi causes pericarditis and pericardial effusion. It can be effectively treated with debridement, drainage, and a prolonged course of antibiotics. In vitro antibiotic susceptibility should be checked as resistance to antibiotics can develop, especially if drainage is inadequate.

Background: Sulfur oxides released from the burning of oil causes severe environmental pollution. The sulfur can be removed via the 4S pathway in biodesulfurization (BDS). Immobilization approaches have been developed to prevent cell contamination of oil during the BDS process. Objectives: The encapsulation of Rhodococcus erythropolis R1 in calcium alginate beads was studied in order to enhance conversion of dibenzothiophene (DBT) to 2-hydroxy biphenyl (2-HBP) as the final product. Also the e...

Nicotine in tobacco is harmful to health and the environment, so there is an environmental requirement to remove nicotine from tobacco and tobacco wastes. In this study, the biotransformation of nicotine by Rhodococcus sp. Y22 was investigated, and three metabolites (NIC1, NIC4 and NIC5) were isolated by column separation, preparative TLC and solid plate's method, respectively. NIC1 was identified as 6-hydoxynicotine based on the results of NMR, MS, HPLC-UV and HRESIMS analysis; NIC4 was a novel compound and identified as 5-(3-methyl-[1,3]oxazinan-2-ylidene)-5H-pyridin-2-one based on the results of NMR, MS and UV analysis; NIC5 was identified as nicotine blue based on the results of NMR and MS analysis. Meanwhile, two metabolites NIC2 and NIC3 were identified as 6-hydroxy-N-methylmyosmine and 6-hydroxypseudooxynicotine by HRESIMS analysis, respectively. According to these metabolites, the possible pathway of nicotine degradation by Rhodococcus sp. Y22 was proposed. The nicotine can be transformed to nicotine blue through two pathways (A and B), and 6-hydroxy-N-methylmyosmine is the key compound, which can be converted to 6-hydroxypseudooxynicotine (pathway A) and 5-(3-methyl-[1,3]oxazinan-2-ylidene)-5H-pyridin-2-one (pathway B), respectively. Moreover, the encoding gene of nicotine dehydrogenase, ndh, was amplified from Rhodococcus sp. Y22, and its transcriptional level could be up-regulated obviously under nicotine induction. Our studies reported the key metabolites and possible biotransformation pathway of nicotine in Rhodococcus sp. Y22, and provided new insights into the microbial metabolism of nicotine.

Rhodococcus equi is a gram positive, intracellular pathogen of foals worldwide. The aim of this study was to determine whether there was an increasing resistance occurring in Rhodococcus equi towards the antibiotics rifampin and erythromycin over a seven year period. The investigation was carried out with the use of E test strips (epsilometers) for rifampin and erythromycin in order to determine the Minimum Inhibitory Concentrations (MIC) values of Rhodococcus equi to these antibiotics. The main results of this study found that the mean MICs were higher for erythromycin than for rifampin for every year analysed apart from 2008. The results highlight that 75 % (6/8) of the mean MICs for erythromycin were above the threshold of susceptibility of 0.5 μg/ml and one of the yearly mean MICs for rifampin (2008) was above the level of ≤ 1 μg/ml. Two soil samples analysed had high MIC values of 2 μg/ml and 3 μg/ml for rifampin and erythromycin respectively. These samples can be said to have acquired resistance as they are above 1 μg/ml. The significance of these findings is that R. equi is already a problematic pathogen to treat and if the bacteria keeps gaining resistance to these antibiotics at rate that has been shown over the last decade, then a new form of treatment will have to be introduced. Further research into the genomics of Rhodococcus equi will, in time, shed more light on possible alternatives such as vaccines or new, more effective antimicrobials.

The mechanical properties of Rhodococcus RC291 were measured using force spectroscopy equipped with a bacterial cell probe. Rhodococcal cells in the late growth stage of development were found to have greater adhesion to a silicon oxide surface than those in the early growth stage. This is because there are more extracellular polymeric substances (EPS) that contain nonspecific binding sites available on the cells of late growth stage. It is found that EPS in the late exponential phase are les...

Full Text Available Higher plant vasculature is characterized by two distinct developmental phases. Initially, a well-defined radial primary pattern is established. In eudicots, this is followed by secondary growth, which involves development of the cambium and is required for efficient water and nutrient transport and wood formation. Regulation of secondary growth involves several phytohormones, and cytokinins have been implicated as key players, particularly in the activation of cell proliferation, but the molecular mechanisms mediating this hormonal control remain unknown. Here we show that the genes encoding the transcription factor AINTEGUMENTA (ANT and the D-type cyclin CYCD3;1 are expressed in the vascular cambium of Arabidopsis roots, respond to cytokinins and are both required for proper root secondary thickening. Cytokinin regulation of ANT and CYCD3 also occurs during secondary thickening of poplar stems, suggesting this represents a conserved regulatory mechanism.

ABSTRACT Higher plant vasculature is characterized by two distinct developmental phases. Initially, a well-defined radial primary pattern is established. In eudicots, this is followed by secondary growth, which involves development of the cambium and is required for efficient water and nutrient transport and wood formation. Regulation of secondary growth involves several phytohormones, and cytokinins have been implicated as key players, particularly in the activation of cell proliferation, but the molecular mechanisms mediating this hormonal control remain unknown. Here we show that the genes encoding the transcription factor AINTEGUMENTA (ANT) and the D-type cyclin CYCD3;1 are expressed in the vascular cambium of Arabidopsis roots, respond to cytokinins and are both required for proper root secondary thickening. Cytokinin regulation of ANT and CYCD3 also occurs during secondary thickening of poplar stems, suggesting this represents a conserved regulatory mechanism. PMID:26340943

Full Text Available In the protonema of Ceratodon purpureus (Hedw. Brid., apical parts of the protonemal filaments (apical cells, initials of protonemal side branches and of gametophore buds proved to be preferential sites of [14C]-leucine incorporation into proteins. In some filaments, a similar preference for [3H]-uridine incorporation into RNA was observed, whereas in others there was a rather uniform distribution of label over all cells. A short (0.5-2 h treatment with cytokinin (N6-2-isopentenyladenine enhanced [14C]-leucine incorporation, without changing the relative distribution of label. No such enhancement, as well as no change in label distribution could be observed in [3H]-uridine incorporation. No direct relationship seems to exist between the early promotion of protein synthesis by cytokinin in the protonema and cytokinin induction of gametophore buds.

In plants, the G2/M control of cell cycle remains an elusive issue as doubts persist about activatory dephosphorylation--in other eukaryotes provided by CDC25 phosphatase and serving as a final all-or-nothing mitosis regulator. We report on the effects of tobacco (Nicotiana tabacum L., cv. Samsun) transformation with fission yeast (Schizosaccharomyces pombe) cdc25 (Spcdc25) on cell characteristics. Transformed cell suspension cultures showed higher dry mass accumulation during the exponential phase and clustered more circular cell phenotypes compared to chains of elongated WT cells. Similar cell parameters, as in the transformants, can be induced in WT by cytokinins. Spcdc25 cells, after cytokinin treatment, showed giant cell clusters and growth inhibition. In addition, Spcdc25 expression led to altered carbohydrate status: increased starch and soluble sugars with higher sucrose:hexoses ratio, inducible in WT by cytokinin treatment. Taken together, the Spcdc25 transformation had a cytokinin-like effect on studied characteristics. However, endogenous cytokinin determination revealed markedly lower cytokinin levels in Spcdc25 transformants. This indicates that the cells sense Spcdc25 expression as an increased cytokinin availability, manifested by changed cell morphology, and in consequence decrease endogenous cytokinin levels. Clearly, the results on cell growth and morphology are consistent with the model of G2/M control including cytokinin-regulated activatory dephosphorylation. Nevertheless, no clear link is obvious between Spcdc25 transformation and carbohydrate status and thus the observed cytokinin-like effect on carbohydrate levels poses a problem. Hence, we propose that Spcdc25-induced higher CDK(s) activity at G2/M generates a signal-modifying carbohydrate metabolism to meet high energy and C demands of forthcoming cell division.

Full Text Available Soybean is an important crop in Brazil. Nonetheless, there are no reports on the use of plant growth regulator potential in relation to this crop in the national literature. To better understand the role of these compounds, a pot experiment was carried out to study effects of GA3 and cytokinin on the vegetative growth of the soybean. GA3 (50 mg L-1 was applied as seed treatment, leaving plants with water application as control. GA3 (100 mg L-1 and cytokinin (30 mg L-1 were sprayed on leaves at the physiological stage V3/V4, and 15 days after, cytokinin (30 mg L-1, also as foliar spray. Seed treatment decreased plant emergence and initial soybean root growth, but as the season progressed, differences in root growth disappeared; plants were shorter, and presented a decrease in the number of nodes, in stem diameter, in leaf area and in dry matter yield. Conversely, foliar application of GA3 led to an increase in plant height, first node height and stem diameter. Leaf area and dry matter production also increased as a result of GA3 foliar application. There was no effect of exogenous gibberellin and cytokinin on the number of soybean leaves, number of stem branches and root dry matter. Joint application of gibberellin and cytokinin tended to inhibit gibberellin effects. Cytokinin applied to leaves during soybean vegetative growth was not effective in modifying any of the evaluated plant growth variables.A soja é uma das principais culturas no Brasil, porém não há relatos do potencial de utilização dos fitorreguladores nessa cultura na literatura nacional. Para saber mais sobre estes compostos, um experimento em vasos foi conduzido para estudar o efeito do GA3 e citocinina (CK sobre o crescimento vegetativo e floração da soja. GA3 (50 mg L-1 foi aplicado como tratamento de sementes. Plantas somente com aplicação de água foram aplicadas como controle. Foram feitas duas aplicações foliares, sendo na primeira aplicado 100 mg L-1 de GA3

Full Text Available Abstract Background Cytokinin is a plant hormone that plays a crucial role in several processes of plant growth and development. In recent years, major breakthroughs have been achieved in the elucidation of the metabolism, the signal perception and transduction, as well as the biological functions of cytokinin. An important activity of cytokinin is the involvement in chloroplast development and function. Although this biological function has already been known for 50 years, the exact mechanisms remain elusive. Results To elucidate the effects of altered endogenous cytokinin content on the structure and function of the chloroplasts, chloroplast subfractions (stroma and thylakoids from transgenic Pssu-ipt and 35S:CKX1 tobacco (Nicotiana tabacum plants with, respectively, elevated and reduced endogenous cytokinin content were analysed using two different 2-DE approaches. Firstly, thykaloids were analysed by blue-native polyacrylamide gel electrophoresis followed by SDS-PAGE (BN/SDS-PAGE. Image analysis of the gel spot pattern thus obtained from thylakoids showed no substantial differences between wild-type and transgenic tobacco plants. Secondly, a quantitative DIGE analysis of CHAPS soluble proteins derived from chloroplast subfractions indicated significant gel spot abundance differences in the stroma fraction. Upon identification by MALDI-TOF/TOF mass spectrometry, these proteins could be assigned to the Calvin-Benson cycle and photoprotective mechanisms. Conclusion Taken together, presented proteomic data reveal that the constitutively altered cytokinin status of transgenic plants does not result in any qualitative changes in either stroma proteins or protein complexes of thylakoid membranes of fully developed chloroplasts, while few but significant quantitative differences are observed in stroma proteins.

Roots are highly plastic and can acclimate to heterogeneous and stressful conditions. However, there is little knowledge of the effect of moisture gradients on the mechanisms controlling root growth orientation and branching, and how this mechanism may help plants to avoid drought responses. The aim of this study was to isolate mutants of Arabidopsis thaliana with altered hydrotropic responses. Here, altered hydrotropic response 1 (ahr1), a semi-dominant allele segregating as a single gene mutation, was characterized. ahr1 directed the growth of its primary root towards the source of higher water availability and developed an extensive root system over time. This phenotype was intensified in the presence of abscisic acid and was not observed if ahr1 seedlings were grown in a water stress medium without a water potential gradient. In normal growth conditions, primary root growth and root branching of ahr1 were indistinguishable from those of the wild type (wt). The altered hydrotropic growth of ahr1 roots was confirmed when the water-rich source was placed at an angle of 45° from the gravity vector. In this system, roots of ahr1 seedlings grew downward and did not display hydrotropism; however, in the presence of cytokinins, they exhibited hydrotropism like those of the wt, indicating that cytokinins play a critical role in root hydrotropism. The ahr1 mutant represents a valuable genetic resource for the study of the effects of cytokinins in the differential growth of hydrotropism and control of lateral root formation during the hydrotropic response. PMID:22442413

This work aimed at identifying a possible role of phytohormones in long-distance (root-shoot) signaling under nitrogen deficiency. Three-months old pineapple plants were transferred from Murashige and Skoog (MS) medium to nitrogen-free MS (-N). During the first 24h on -N, 20 plants were harvested every 4h. After 30 days in -N, the remaining plants were transferred back to regular MS (+N) and 20 plants harvested every 4h for the first 24h. Following the harvests, endogenous levels of nitrate (NO(3)(-)), indole-3-acetic acid (IAA), isopentenyladenine (iP), isopentenyladenine riboside (iPR), zeatin (Z) and zeatin riboside (ZR) were analyzed in roots and leaves. In N-starved plants, the NO(3)(-) level dropped by 20% in roots between the first (4h) and the second harvest (8h). In leaves a reduction of 20% was found 4h later. Accumulation of IAA peaked in leaves at 16h. In roots, the accumulation of IAA only started at 16h while the leaf content was already in decline, which suggests that the hormone might have traveled from the leaves to the roots, communicating N-shortage. The contents of the four cytokinins were generally low in both, shoot and roots, and remained almost unchanged during the 24h of analysis. After N re-supply, roots showed a NO(3)(-) peak at 8h whereas the foliar concentration increased 4h later. Hormone levels in roots climaxed at 8h, this coinciding with the highest NO(3)(-) concentration. In leaf tissue, a dramatic accumulation was only observed for Z and ZR, and the peak was seen 4h later than in roots, suggesting that Z-type cytokinins might have traveled from the roots to the leaves. These findings provide evidence that there is a signaling pathway for N availability in pineapple plants, communicated upwards through cytokinins (N-supplemented plants) and downwards through auxin (N-starved plants).

The initial rates of disappearance of cytokinins, as determined by high pressure liquid chromatography, for tomatoes which were vacuum infused with benzyladenine and isopentenyladenine were dissimilar between the normal ripening (Ohio CR-6 and Rutgers), ripening inhibited mutant (RIN) and non-ripening mutant (NOR) tomato varieties. Radiolabeled (8-/sup 14/C)Benzyladenine metabolism was followed during a 2 h period utilizing thin layer chromatography and visualization by fluorography. The (8-/sup 14/C)Benzyladenine metabolite patterns were different among the varieties. The (8-/sup 14/C)Benzyladenine metabolite pattern in Ohio CR-6 tomato changed as the fruit ripened.

Full Text Available Induction of adventitious shoot formation from mature zygotic embryo of oil palm was carried out in liquid MS mediumsupplemented with various types of cytokinins. Kinetin (KN alone at concentration of 0.5 mg/l gave the highest adventitiousshoot formation at 13.4%. However, abnormal shoots in form of inflorescence-like structure (ILS were obtained in 5mg/l KN containing medium. For coconut water a big ILSs were formed at 10.6%. Histological studied revealed that thoseinflorescences had no clear floral organs.

Protocol was developed for acceleration of microtuber induction in potato. It was observed that cytokinine (Kin), and both dark and light period had great influence on in vitro tuberization. Regarding concentration of Kin, 8 mg/l was found excellent for microtuber induction and formation under both dark and light conditions. Under continuous dark condition, higher percentage of explants (shoot) induced microtuber was observed than the short and long period of light conditions. For increasing the number and weight of microtuber product/explant, use of long photoperiod (16 h) is recommended.

Full Text Available Water-deficit stress is a major environmental factor that limits agricultural productivity worldwide. Recent episodes of extreme drought have severely affected cotton production in the Southwestern USA. There is a pressing need to develop cotton varieties with improved tolerance to water-deficit stress for sustainable production in water-limited regions. One approach to engineer drought tolerance is by delaying drought-induced senescence via up-regulation of cytokinin biosynthesis. The isopentenyltransferase gene (IPT that encodes a rate limiting enzyme in cytokinin biosynthesis, under the control of a water-deficit responsive and maturation specific promoter P(SARK was introduced into cotton and the performance of the P(SARK::IPT transgenic cotton plants was analyzed in the greenhouse and growth chamber conditions. The data indicate that P(SARK::IPT-transgenic cotton plants displayed delayed senescence under water deficit conditions in the greenhouse. These plants produced more root and shoot biomass, dropped fewer flowers, maintained higher chlorophyll content, and higher photosynthetic rates under reduced irrigation conditions in comparison to wild-type and segregated non-transgenic lines. Furthermore, P(SARK::IPT-transgenic cotton plants grown in growth chamber condition also displayed greater drought tolerance. These results indicate that water-deficit induced expression of an isopentenyltransferase gene in cotton could significantly improve drought tolerance.

Chromatin architecture determines transcriptional accessibility to DNA and consequently gene expression levels in response to developmental and environmental stimuli. Recently, chromatin remodelers such as SWI/SNF complexes have been recognized as key regulators of chromatin architecture. To gain insight into the function of these complexes during root development, we have analyzed Arabidopsis knock-down lines for one sub-unit of SWI/SNF complexes: BAF60. Here, we show that BAF60 is a positive regulator of root development and cell cycle progression in the root meristem via its ability to down-regulate cytokinin production. By opposing both the deposition of active histone marks and the formation of a chromatin regulatory loop, BAF60 negatively regulates two crucial target genes for cytokinin biosynthesis (IPT3 and IPT7) and one cell cycle inhibitor (KRP7). Our results demonstrate that SWI/SNF complexes containing BAF60 are key factors governing the equilibrium between formation and dissociation of a chromatin loop controlling phytohormone production and cell cycle progression.

The root serves as an essential organ in plant growth by taking up nutrients and water from the soil and supporting the rest of the plant body. Some plant species utilize roots as storage organs. Sweet potatoes (Ipomoea batatas), cassava (Manihot esculenta), and radish (Raphanus sativus), for example, are important root crops. However, how their root growth is regulated remains unknown. In this study, we characterized the relationship between cambium and radial root growth in radish. Through a comparative analysis with Arabidopsis root expression data, we identified putative cambium-enriched transcription factors in radish and analysed their expression in representative inbred lines featuring distinctive radial growth. We found that cell proliferation activities in the cambium positively correlated with radial growth and final yields of radish roots. Expression analysis of candidate transcription factor genes revealed that some genes are differentially expressed between inbred lines and that the difference is due to the distinct cytokinin response. Taken together, we have demonstrated for the first time, to the best of our knowledge, that cytokinin-dependent radial growth plays a key role in the yields of root crops.

Cytokinins and gibberellins (GAs) play antagonistic roles in regulating reproductive meristem activity. Cytokinins have positive effects on meristem activity and maintenance. During inflorescence meristem development, cytokinin biosynthesis is activated via a KNOX-mediated pathway. Increased cytokinin activity leads to higher grain number, whereas GAs negatively affect meristem activity. The GA biosynthesis genes GA20oxs are negatively regulated by KNOX proteins. KNOX proteins function as modulators, balancing cytokinin and GA activity in the meristem. However, little is known about the crosstalk among cytokinin and GA regulators together with KNOX proteins and how KNOX-mediated dynamic balancing of hormonal activity functions. Through map-based cloning of QTLs, we cloned a GA biosynthesis gene, Grain Number per Panicle1 (GNP1), which encodes rice GA20ox1. The grain number and yield of NIL-GNP1TQ were significantly higher than those of isogenic control (Lemont). Sequence variations in its promoter region increased the levels of GNP1 transcripts, which were enriched in the apical regions of inflorescence meristems in NIL-GNP1TQ. We propose that cytokinin activity increased due to a KNOX-mediated transcriptional feedback loop resulting from the higher GNP1 transcript levels, in turn leading to increased expression of the GA catabolism genes GA2oxs and reduced GA1 and GA3 accumulation. This rebalancing process increased cytokinin activity, thereby increasing grain number and grain yield in rice. These findings uncover important, novel roles of GAs in rice florescence meristem development and provide new insights into the crosstalk between cytokinin and GA underlying development process. PMID:27764111

Cytokinins and gibberellins (GAs) play antagonistic roles in regulating reproductive meristem activity. Cytokinins have positive effects on meristem activity and maintenance. During inflorescence meristem development, cytokinin biosynthesis is activated via a KNOX-mediated pathway. Increased cytokinin activity leads to higher grain number, whereas GAs negatively affect meristem activity. The GA biosynthesis genes GA20oxs are negatively regulated by KNOX proteins. KNOX proteins function as modulators, balancing cytokinin and GA activity in the meristem. However, little is known about the crosstalk among cytokinin and GA regulators together with KNOX proteins and how KNOX-mediated dynamic balancing of hormonal activity functions. Through map-based cloning of QTLs, we cloned a GA biosynthesis gene, Grain Number per Panicle1 (GNP1), which encodes rice GA20ox1. The grain number and yield of NIL-GNP1TQ were significantly higher than those of isogenic control (Lemont). Sequence variations in its promoter region increased the levels of GNP1 transcripts, which were enriched in the apical regions of inflorescence meristems in NIL-GNP1TQ. We propose that cytokinin activity increased due to a KNOX-mediated transcriptional feedback loop resulting from the higher GNP1 transcript levels, in turn leading to increased expression of the GA catabolism genes GA2oxs and reduced GA1 and GA3 accumulation. This rebalancing process increased cytokinin activity, thereby increasing grain number and grain yield in rice. These findings uncover important, novel roles of GAs in rice florescence meristem development and provide new insights into the crosstalk between cytokinin and GA underlying development process.

The present paper introduces a highly sensitive and selective method for simultaneous quantification of 12 cytokinins (free form and their conjugates). The method includes a protocol of extraction with methanol/water/formic acid (15/4/1, v/v/v) to the micro-scale samples, pre-purification with solid phase extraction (SPE) cartridges of the extracts, separation with a high performance liquid chromatography (HPLC) and detection by an electrospray ionization ion trap mass spectrometry (ESI-Ion trap-MS) system in a consecutive ion monitoring (CRM) mode at the three stage fragmentation of mass spectrometry (MS(3) ). The lowest detection level of the cytokinins of the method reaches 0.1-2.0 pg with a very wide range of linear regression from 1-512 pg, at the coefficient factors of 0.98-0.99. The feasibility of this method has been proven in the application of the method to the analysis of the trace-amount contents of cytokinins in the micro-scale samples of various types of plant materials, such as aerial parts of rice and poplar leaves etc. 12 endogenous cytokinins had been identified and quantified in the plant tissues, with an acceptable relatively higher recovery rate from 40% to 70%.

The present paper introduces a highly sensitive and selective method for simultaneous quantification of 12 cytokinins(free form and their conjugates).The method includes a protocol of extraction with methanol/water/formic acid(1514/1,v/v/v)to the micro-scale samples,pre-purification with solid phase extraction(SPE)cartridges of the extracts,separation with a high performance liquid chromatography(HPLC)and detection by an electrospray ionization ion trap mass spectrometry(ESI-Ion trap-MS)system in a consecutive ion monitoring(CRM)mode at the three stage fragmentation of mass spectrometry(MS3).The lowest detection level of the cytokinins of the method reaches 0.1-2.0 pg with a very wide range of linear regression from 1-512 pg,at the coefficient factors of 0.98-0.99.The feasibility of this method has been proven in the application of the method to the analysis of the trace-amount contents of cytokinins in the micro-scale samples of various types of plant materials,such as aerial parts of rice and poplar leaves etc.12 endogenous cytokinins had been identified and quantified in the plant tissues,with an acceptable relatively higher recovery rate from 40% to 70%.

Cytokinins (CKs) are well-established as important phytohormonal regulators of plant growth and development. An increasing number of studies have also revealed the function of these hormones in plant responses to biotic and abiotic stresses. While the function of certain CK classes, including tra...

Plants of an inbred line of Plantago major ssp. pleiosperma were subjected to an alteration in mineral supply. Observed responses of growth rate and shoot to root ratio are thought to be induced by changes in endogenous cytokinin concentration and not by mineral concentration in plant tissue.

Legumes host their rhizobium symbiont in novel root organs, called nodules. Nodules originate from differentiated root cortical cells that de-differentiate and subsequently form nodule primordia, a process controlled by cytokinin. A whole genome duplication (WGD) has occurred at the root of the legu

Initiation of symbiotic nodules in legumes requires cytokinin signaling, but its mechanism of action is largely unknown. Here, we tested whether the failure to initiate nodules in the Medicago truncatula cytokinin perception mutant cre1 (cytokinin response1) is due to its altered ability to regulate auxin transport, auxin accumulation, and induction of flavonoids. We found that in the cre1 mutant, symbiotic rhizobia cannot locally alter acro- and basipetal auxin transport during nodule initiation and that these mutants show reduced auxin (indole-3-acetic acid) accumulation and auxin responses compared with the wild type. Quantification of flavonoids, which can act as endogenous auxin transport inhibitors, showed a deficiency in the induction of free naringenin, isoliquiritigenin, quercetin, and hesperetin in cre1 roots compared with wild-type roots 24 h after inoculation with rhizobia. Coinoculation of roots with rhizobia and the flavonoids naringenin, isoliquiritigenin, and kaempferol, or with the synthetic auxin transport inhibitor 2,3,5,-triiodobenzoic acid, rescued nodulation efficiency in cre1 mutants and allowed auxin transport control in response to rhizobia. Our results suggest that CRE1-dependent cytokinin signaling leads to nodule initiation through the regulation of flavonoid accumulation required for local alteration of polar auxin transport and subsequent auxin accumulation in cortical cells during the early stages of nodulation.

Cytokinin, auxin and gibberellin content in resting and wound-responding potato tuber are not clearly defined. Consequently, the coordination and possible networking of these classical hormones in the regulation of wound-healing processes are poorly understood. Using a well-defined tuber wound-hea...

Cassava (Manihot esculenta Crantz) sheds its leaves during growth, especially within the tropical dry season. With the production of SAG12-IPT transgenic cassava we want to test the level of leaf retention and altered cytokinin metabolism of transgenic plants via the autoregulatory senescence inhibition system. After confirmation of transgene expression by molecular analysis and phenotype examination in greenhouse plants, two transgenic plant lines, 529-28 and 529-48, were chosen for further investigation. Detached mature leaves of 529-28 plants retained high levels of chlorophyll compared with wild-type leaves after dark-induced senescence treatment. Line 529-28 showed significant drought tolerance as indicated by stay-green capacity after drought stress treatment. Field experiments proved that leaf senescence syndrome was significantly delayed in 529-28 plants in comparison with wild-type and 529-48 plants. Physiological and agronomical characterizations of these plants also revealed that the induced expression of IPT had effects on photosynthesis, sugar allocation and nitrogen partitioning. Importantly, the 529-28 plants accumulated a high level of trans-zeatin-type cytokinins particularly of corresponding storage O-glucosides to maintain cytokinin homeostasis. Our study proves the feasibility of prolonging the leaf life of woody cassava and also sheds light on the control of cytokinin homeostasis in cassava leaves.

Legumes host their rhizobium symbiont in novel root organs, called nodules. Nodules originate from differentiated root cortical cells that de-differentiate and subsequently form nodule primordia, a process controlled by cytokinin. A whole genome duplication (WGD) has occurred at the root of the

Lignocellulosic biomass can be a significant source of renewable clean energy with continued improvement in biomass yield and bioconversion strategies. In higher plants, the leaf blade is the central energy convertor where solar energy and CO2 are assimilated to make the building blocks for biomass production. Here we report that introducing the leaf blade development regulator STENOFOLIA (STF), a WOX family transcription factor, into the biofuel crop switchgrass, significantly improves both biomass yield and sugar release. We found that STF overexpressing switchgrass plants produced approximately 2-fold more dry biomass and release approximately 1.8-fold more solubilized sugars without pretreatment compared to controls. The biomass increase was attributed mainly to increased leaf width and stem thickness, which was also consistent in STF transgenic rice and Brachypodium, and appeared to be caused by enhanced cell proliferation. STF directly binds to multiple regions in the promoters of some cytokinin oxidase/dehydrogenase (CKX) genes and represses their expression in all three transgenic grasses. This repression was accompanied by a significant increase in active cytokinin content in transgenic rice leaves, suggesting that the increase in biomass productivity and sugar release could at least in part be associated with improved cytokinin levels caused by repression of cytokinin degrading enzymes. Our study provides a new tool for improving biomass feedstock yield in bioenergy crops, and uncovers a novel mechanistic insight in the function of STF, which may also apply to other repressive WOX genes that are master regulators of several key plant developmental programs. PMID:28264034

Lignocellulosic biomass can be a significant source of renewable clean energy with continued improvement in biomass yield and bioconversion strategies. In higher plants, the leaf blade is the central energy convertor where solar energy and CO2 are assimilated to make the building blocks for biomass production. Here we report that introducing the leaf blade development regulator STENOFOLIA (STF), a WOX family transcription factor, into the biofuel crop switchgrass, significantly improves both biomass yield and sugar release. We found that STF overexpressing switchgrass plants produced approximately 2-fold more dry biomass and release approximately 1.8-fold more solubilized sugars without pretreatment compared to controls. The biomass increase was attributed mainly to increased leaf width and stem thickness, which was also consistent in STF transgenic rice and Brachypodium, and appeared to be caused by enhanced cell proliferation. STF directly binds to multiple regions in the promoters of some cytokinin oxidase/dehydrogenase (CKX) genes and represses their expression in all three transgenic grasses. This repression was accompanied by a significant increase in active cytokinin content in transgenic rice leaves, suggesting that the increase in biomass productivity and sugar release could at least in part be associated with improved cytokinin levels caused by repression of cytokinin degrading enzymes. Our study provides a new tool for improving biomass feedstock yield in bioenergy crops, and uncovers a novel mechanistic insight in the function of STF, which may also apply to other repressive WOX genes that are master regulators of several key plant developmental programs.

A new, naturally occurring cytokinin was isolated from a cell culture of anise (Pimpinella anisum L.). Its structure was determined as 6-benzylaminopurine riboside by the use of gas chromatography and mass spectrometry. Quantitation of this new plant hormone was accomplished by single-ion-monitoring measurements.

To get a broader view on the molecular mechanisms underlying somatic embryogenesis (SE) in cotton (Gossypium hirsutum L.),global analysis of cotton transcriptome dynamics during SE in different sister lines was performed using RNA-Seq.A total of 204 349 unigenes were detected by de novo assembly of the 214 977 462 Illumina reads.The quantitative reverse transcription-polymerase chain reaction (qRT-PCR) measurements were positively correlated with the RNA-Seq results for almost all the tested genes (R2 =0.841,correlation was significant at the 0.01 level).Different phytohormone (auxin and cytokinin) concentration ratios in medium and the endogenous content changes of these two phytohormones at two stages in different sister lines suggested the roles of auxin and cytokinin during cotton SE.On the basis of global gene regulation of phytohormone-related genes,numerous genes from all the differentially expressed transcripts were involved in auxin and cytokinin biosynthesis and signal transduction pathways.Analyses of differentially expressed genes that were involved in these pathways revealed the substantial changes in gene type and abundance between two sister lines.Isolation,cloning and silencing/ overexpressing the genes that revealed remarkable up-or down-expression during cotton SE were important.Furthermore,auxin and cytokinin play a primary role in SE,but potential cross-talk with each other or other factors remains unclear.

A Rhodococcus sp. UKMP-5M isolate was shown to detoxify cyanide successfully, suggesting the presence of an intrinsic property in the bacterium which required no prior cyanide exposure for induction of this property. However, in order to promote growth, Rhodococcus sp. UKMP-5M was fully acclimatized to cyanide after 7 successive subcultures in 0.1 mM KCN for 30 days. To further shorten the lag phase and simultaneously increase the tolerance towards higher cyanide concentrations, the bacterium was induced with various nitrile compounds sharing a similar degradatory pathway to cyanide. Acetonitrile emerged as the most favored inducer and the induced cells were able to degrade 0.1 mM KCN almost completely within 18 h. With the addition of subsequent aliquots of 0.1 mM KCN a shorter period for complete removal of cyanide was required, which proved to be advantageous economically. Both resting cells and crude enzyme of Rhodococcus sp. UKMP-5M were able to biodegrade cyanide to ammonia and formate without the formation of formamide, implying the identification of a simple hydrolytic cyanide degradation pathway involving the enzyme cyanidase. Further verification with SDS-PAGE revealed that the molecular weight of the active enzyme was estimated to be 38 kDa, which is consistent with previously reported cyanidases. Since the recent advancement in the application of biological methods in treating cyanide-bearing wastewater has been promising, the discovery of this new bacterium will add value by diversifying the existing microbial populations capable of cyanide detoxification.

The psychrotroph Rhodococcus sp. strain Q15 was examined for its ability to degrade individual n-alkanes and diesel fuel at low temperatures, and its alkane catabolic pathway was investigated by biochemical and genetic techniques. At 0 and 5 C, Q15 mineralized the short-chain alkanes dodecane and hexadecane to a greater extent than that observed for the long-chain alkanes octacosane and dotriacontane. Q15 utilized a broad range of aliphatics (C{sub 10} to C{sub 21} alkanes, branched alkanes, and a substituted cyclohexane) present in diesel fuel at 5 C. Mineralization of hexadecane at 5 C was significantly greater in both hydrocarbon-contaminated and pristine soil microcosms seeded with Q15 cells than in uninoculated control soil microcosms. The detection of hexadecane and dodecane metabolic intermediates (1-hexadecanol and 2-hexadecanol and 1-do-decanol and 2-dodecanone, respectively) by solid-phase microextraction-gas chromatography-mass spectrometry and the utilization of potential metabolic intermediates indicated that Q15 oxidizes alkanes by both the terminal oxidation pathway and the subterminal oxidation pathway. Genetic characterization by PCR and nucleotide sequence analysis indicated that Q15 possesses an aliphatic aldehyde dehydrogenase gene highly homologous to the Rhodococcus erythropolis thcA gene. Rhodococcus sp. strain Q15 possessed two large plasmids of approximately 90 and 115 kb (shown to mediate Cd resistance) which were not required for alkane mineralization, although the 90-kb plasmid enhanced mineralization of some alkanes and growth on diesel oil at both 5 and 25 C.

The efficiency of inorganic nitrogen (N) assimilation is a critical component of fertilizer use by plants and of forage production in Lolium perenne, an important pasture species worldwide. We present a spatiotemporal description of nitrate use efficiency in terms of metabolic responses and carbohydrate remobilization, together with components of cytokinin signal transduction following nitrate addition to N-impoverished plants. Perennial ryegrass (L. perenne cv. Grasslands Nui) plants were grown for 10 weeks in unfertilized soil and then treated with nitrate (5 mM) hydroponically. Metabolomic analysis by gas chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry revealed a dynamic interaction between N and carbon metabolism over a week-long time course represented by the relative abundance of amino acids, tricarboxylic acid intermediates and stored water-soluble carbohydrates (WSCs). The initial response to N addition was characterized by a rapid remobilization of carbon stores from the low-molecular weight WSC, along with an increase in N content and assimilation into free amino acids. Subsequently, the shoot became the main source of carbon through remobilization of a large pool of high-molecular weight WSC. Associated quantification of cytokinin levels and expression profiling of putative cytokinin response regulator genes by quantitative reverse transcription polymerase chain reaction support a role for cytokinin in the mediation of the response to N addition in perennial ryegrass. The presence of high levels of cis-zeatin-type cytokinins is discussed in the context of hormonal homeostasis under the stress of steady-state N deficiency.

Zearalenone (hereafter referred to as ZEA) is a nonsteroidal estrogenic mycotoxin produced by several Fusarium spp. on cereal grains. ZEA is one of the most hazardous natural endocrine disrupting chemicals (EDC) which induces hyper estrogenic responses in mammals. This can result in reproductive disorders in farm animals as well as in humans. Consequently, detoxification strategies for contaminated crops are crucial for food safety. In this study we have developed a bacterial based detoxification system using a non-pathogen Rhodococcus pyridinivorans K408 strain. Following 5 days treatment of ZEA with R. pyridinivorans K408 strain HPLC analyses showed an 87.21% ZEA-degradation efficiency of the bacterial enzyme systems. In another approach, the strain biotransformation ability has also been confirmed by a bioluminescent version of the yeast estrogen screening system (BLYES), which detected an 81.75% of biodegradability of ZEA, in a good agreement with the chemical analyses. Furthermore, the capacity of R. pyridinivorans to eliminate the estrogenic effects of ZEA was tested by using an immature uterotrophic assay. Prepubertal female rats were treated with vehicle (olive oil), 17β-estradiol, ZEA (0.1-1-5-10 mg/kg body weight) and LB broth containing 500 mg/l ZEA that has already been incubated with or without Rhodococcus pyridinivorans K408 strain. Uterine weights were measured and the mRNA level changes relating to apelin, aquaporin 5, complement component 2, and calbindin-3 genes were measured by qRT-PCR. These genes represent the major pathways that are affected by estromimetic compounds. Zearalenone feeding significantly increased the uterus weight in a dose dependent manner and at the same time upregulated complement component 2 and calbindin-3 expression as well as decreased apelin and aquaporin 5 mRNA levels comparable to that seen in 17β-estradiol exposed rats. In contrast, LB broth in which ZEA was incubated with Rhodococcus pyridinivorans K408 prior to

The 1,3-dinitrobenzene-degrading Rhodococcus strain QT-1 was isolated under nitrogen limiting conditions from contaminated soil samples. Experimental data indicate tha 1,3-dinitrobenzene is metabolized via 4-nitrocatechol. Both compounds were oxidized by resting cells and nitro groups were completely eliminated as nitrite. Strain QT-1 utilizes both 1,3-dinitrobenzene and 4-nitrocatechol as source of nitrogen in the absence as well as in the presence of high amounts of ammonia. Growth on 4-nitrocatechol does not induce the enzyme(s) for the initial oxidation of 1,3-dinitrobenzene.

We describe sequence and gene organization of a new glycopeptide resistance operon (vanO) in Rhodococcus equi from soil. The vanO operon has low homology to enterococccal van operons and harbors a vanHOX cluster transcribed in opposite direction to the vanS-vanR regulatory system and comprised...... between three open reading frames with unknown function. This finding has clinical interest since glycopeptides are used to treat R. equi infections and resistance has been reported in clinical isolates....

Abstract Rhodococcus equi is an unusual cause of infection in humans, but has emerged as an opportunistic pathogen among immunocompromised patients. Primary pulmonary involvement is the most common clinical presentation, although the spectrum of disease is broad. Diagnosing R. equi infections remains challenging, both from clinical and microbiological view, and no standard treatment has been established. In this report, we present a detailed case of a 57-year-old male renal transplant recipient who developed R. equi bacteremia with a concomitant Pneumocystis jirovecii pneumonia. We describe the clinical features of R. equi infections, highlight the importance of an early diagnosis, and briefly review treatment options for this rare infection. PMID:27500216

Full Text Available The authors reported a lung infection by Rhodococcus equi in a 25 years-old male patient admitted to hospital with cough, dyspnea, fever, and previous diagnosis of pleural effusion. R. equi was isolated from pleural fluid and the patient acquired nosocomial infection by Acinetobacter baumannii, isolated from chest drain. The patient was treated with antibiotics. During hospitalization, he was diagnosed with non-Hodgkin lymphoma of precursor T-cell lymphoblastic lymphoma subtype in biopsy of pleura. After undergoing surgery for pulmonary decortication for drain empyema, the patient died due to septicemia.

The psychrotroph Rhodococcus sp. strain Q15 was examined for its ability to degrade individual n-alkanes and diesel fuel at low temperatures, and its alkane catabolic pathway was investigated by biochemical and genetic techniques. At 0 and 5°C, Q15 mineralized the short-chain alkanes dodecane and hexadecane to a greater extent than that observed for the long-chain alkanes octacosane and dotriacontane. Q15 utilized a broad range of aliphatics (C10 to C21 alkanes, branched alkanes, and a substi...

Cytokinins (CKs) are an important group of phytohormones. Their tightly regulated and balanced levels are essential for proper cell division and plant organ development. Here we report precise quantification of CK metabolites and other phytohormones in maize reproductive organs in the course of pollination and kernel maturation. A novel enzymatic activity dependent on NADP(+) converting trans-zeatin (tZ) to 6-(3-methylpyrrol-1-yl)purine (MPP) was detected. MPP shows weak anticytokinin properties and inhibition of CK dehydrogenases due to their ability to bind to an active site in the opposite orientation than substrates. Although the physiological significance of tZ side-chain cyclization is not anticipated as the MPP occurrence in maize tissue is very low, properties of the novel CK metabolite indicate its potential for utilization in plant in vitro tissue culture. Furthermore, feeding experiments with different isoprenoid CKs revealed distinct preferences in glycosylation of tZ and cis-zeatin (cZ). While tZ is preferentially glucosylated at the N9 position, cZ forms mainly O-glucosides. Since O-glucosides, in contrast to N9-glucosides, are resistant to irreversible cleavage catalyzed by CK dehydrogenases, the observed preference of maize CK glycosyltransferases to O-glycosylate zeatin in the cis-position might be a reason why cZ derivatives are over-accumulated in different maize tissues and organs.

Rhodococcus species have become increasingly important owing to their ability to degrade a wide range of toxic chemicals and produce bioactive compounds. Here, we report isolation of the Rhodococcus sp. KB6, which is a new leaf-inhabiting endophytic bacterium that suppresses black rot disease in sweet potato leaves. We determined the 7.0 Mb draft genome sequence of KB6 and have predicted 19 biosynthetic gene clusters for secondary metabolites, including heterobactins, which are a new class of siderophores. Notably, we showed the first internal colonization of host plants with Rhodococcus sp. KB6 and discuss its potential as a biocontrol agent for sustainable agriculture.

The phytohormones cytokinin and auxin orchestrate the root meristem development in angiosperms by determining embryonic bipolarity. Ferns, having the most basal euphyllophyte root, form neither bipolar embryos nor permanent embryonic primary roots but rather an adventitious root system. This raises

The phytohormones cytokinin and auxin orchestrate the root meristem development in angiosperms by determining embryonic bipolarity. Ferns, having the most basal euphyllophyte root, form neither bipolar embryos nor permanent embryonic primary roots but rather an adventitious root system. This raises

A monoterpene ε-lactone hydrolase (MLH) from Rhodococcus erythropolis DCL14, catalyzing the ring opening of lactones which are formed during degradation of several monocyclic monoterpenes, including carvone and menthol, was purified to apparent homogeneity. It is a monomeric enzyme of 31 kDa that is

A strain of Rhodococcus designated MB1, which was capable of utilizing cocaine as a sole source of carbon and nitrogen for growth, was isolated from rhizosphere soil of the tropane alkaloid-producing plant Erythroxylum coca. A cocaine esterase was found to initiate degradation of cocaine, which was hydrolyzed to ecgonine methyl ester and benzoate; both of these esterolytic products were further metabolized by Rhodococcus sp. strain MB1. The structural gene encoding a cocaine esterase, designated cocE, was cloned from Rhodococcus sp. strain MB1 genomic libraries by screening recombinant strains of Rhodococcus erythropolis CW25 for growth on cocaine. The nucleotide sequence of cocE corresponded to an open reading frame of 1,724 bp that codes for a protein of 574 amino acids. The amino acid sequence of cocaine esterase has a region of similarity with the active serine consensus of X-prolyl dipeptidyl aminopeptidases, suggesting that the cocaine esterase is a serine esterase. The cocE coding sequence was subcloned into the pCFX1 expression plasmid and expressed in Escherichia coli. The recombinant cocaine esterase was purified to apparent homogeneity and was found to be monomeric, with an M(r) of approximately 65,000. The apparent K(m) of the enzyme (mean +/- standard deviation) for cocaine was measured as 1.33 +/- 0.085 mM. These findings are of potential use in the development of a linked assay for the detection of illicit cocaine.

The explosive Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is known to be degraded aerobically by various isolates of the Rhodococcus species, with denitration being the key step, mediated by Cytochrome P450. Our study aimed at gaining insight into the RDX degradation mechanism by Rhodococcus species and comparing isotope effects associated with RDX degradation by distinct Rhodococcus strains. For these purposes, enrichment in (13)C and (15)N isotopes throughout RDX denitration was studied for three distinct Rhodococcus strains, isolated from soil and groundwater in an RDX-contaminated site. The observable (15)N enrichment throughout the reaction, together with minor (13)C enrichment, suggests that N-N bond cleavage is likely to be the key rate-limiting step in the reaction. The similarity in the kinetic (15)N isotope effect between the three tested strains suggests that either isotope-masking effects are negligible, or are of a similar extent for all tested strains. The lack of variability in the kinetic (15)N isotope effect allows the interpretation of environmental studies with greater confidence.

The structure of the alcohol dehydrogenase ADH-'A' from Rhodococcus ruber reveals possible reasons for its remarkable tolerance to organic co-solvents and suggests new directions for structure-informed mutagenesis to produce enzymes of altered substrate specificity or improved selectivity.

Rhodococcus erythropolis PR4 is an alkane-degrading bacterium, which grows well in media containing high concentrations of alkanes. These properties give the organism potential in the bioremediation of various environments contaminated by alkanes. In this study, we report the translocation of R. ery

Rhodococcus equi causes fatal pyogranulomatous pneumonia in foals and immunocompromised animals and humans. Despite its importance, there is currently no effective vaccine against the disease. The actinobacteria R. equi and the human pathogen Mycobacterium tuberculosis are related, and both cause pu

A monoterpene ε-lactone hydrolase (MLH) from Rhodococcus erythropolis DCL14, catalyzing the ring opening of lactones which are formed during degradation of several monocyclic monoterpenes, including carvone and menthol, was purified to apparent homogeneity. It is a monomeric enzyme of 31 kDa that is

Full Text Available In this study, benzoate dioxygenase from Rhodococcus ruber UKMP-5M was catalyzed by oxidating the benzene ring to catechol and other derivatives. The benzoate dioxygenase (benA gene from Rhodococcus ruber UKMP-5M was then expressed, purified, characterized, The benA gene was amplified (642 bp, and the product was cloned into a pGEM-T vector.The recombinant plasmid pGEMT-benA was digested by double restriction enzymes BamHI and HindIII to construct plasmid pET28b-benA and was then ligated into Escherichia coli BL21 (DE3. The recombinant E. coli was induced with 0.5 mM isopropyl β-D-thiogalactoside (IPTG at 22˚C to produce benzoate dioxygenase. The enzyme was then purified by ion exchange chromatography after 8 purification folds. The resulting product was 25 kDa, determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE and western blotting. Benzoate dioxygenase activity was found to be 6.54 U/mL and the optimal pH and temperature were 8.5 and 25°C, respectively. Maximum velocity (Vmax and Michaelis constant (Km were 7.36 U/mL and 5.58 µM, respectively. The end metabolite from the benzoate dioxygenase reaction was cyclohexane dione, which was determined by gas chromatography mass spectrometry (GC-MS.

The mechanism of transport of (14)C-fluoranthene by Rhodococcus sp. BAP-1, a Gram-positive bacterium isolated from crude oil-polluted soil, was examined. Our finding demonstrated that the mechanism for fluoranthene travel across the cell membrane in Rhodococcus sp. BAP-1 requires energy. Meanwhile, the transport of fluoranthene involves concurrent catabolism of (14)C, that leading to the generation of significant amount of (14)CO2. Combined with trans-membrane transport dynamic and response surface methodology, a significant influence of temperature, pH and salinity on cellular uptake rate was screened by Plackett-Burman design. Then, Box-Behnken design was employed to optimize and enhanced the trans-membrane transport process. The results predicted by Box-Behnken design indicated that the maximum cellular uptake rate of fluoranthene could be achieve to 0.308μmolmin(-1)mg(-1)·protein (observed) and 0.304μmolmin(-1)mg(-1)·protein (predicted) when the initial temperature, pH and salinity were set at 20°C, 9% and 1%, respectively.

While applications of amine oxidases are increasing, few have been characterised and our understanding of their biological role and strategies for bacteria exploitation are limited. By altering the nitrogen source (NH4Cl, putrescine and cadaverine (diamines) and butylamine (monoamine)) and concentration, we have identified a constitutive flavin dependent oxidase (EC 1.4.3.10) within Rhodococcus opacus. The activity of this oxidase can be increased by over two orders of magnitude in the presence of aliphatic diamines. In addition, the expression of a copper dependent diamine oxidase (EC 1.4.3.22) was observed at diamine concentrations>1mM or when cells were grown with butylamine, which acts to inhibit the flavin oxidase. A Michaelis-Menten kinetic treatment of the flavin oxidase delivered a Michaelis constant (KM)=190μM and maximum rate (kcat)=21.8s(-1) for the oxidative deamination of putrescine with a lower KM (=60μM) and comparable kcat (=18.2s(-1)) for the copper oxidase. MALDI-TOF and genomic analyses have indicated a metabolic clustering of functionally related genes. From a consideration of amine oxidase specificity and sequence homology, we propose a putrescine degradation pathway within Rhodococcus that utilises oxidases in tandem with subsequent dehydrogenase and transaminase enzymes. The implications of PUT homeostasis through the action of the two oxidases are discussed with respect to stressors, evolution and application in microbe-assisted phytoremediation or bio-augmentation.

A new aliphatic amidase gene (ami), having a level of similarity with the nearest homologs of no more than 77%, was identified in the Rhodococcus erythropolis TA37 strain, which is able to hydrolyze a wide range of amides. The amidase gene was cloned within a 3.7 kb chromosomal locus, which also contains putative acetyl-CoA ligase and ABC-type transportergenes. The structure of this locus in the R. erythropolis TA37 strain differs from the structure of loci in other Rhodococcus strains. The amidase gene is expressed in Escherichia coli cells. It was demonstrated that amidase (generated in the recombinant strain) efficiently hydrolyzes acetamide (aliphatic anmide) and does not use 4'-nitroacetanilide (N-substituted amide) as a substrate. Insertional inactivation of the amidase gene in the R. erythropolis TA37 strain results in a considerable decrease (by at least 6-7 times) in basal amidase activity, indicating functional amidase activity in the R. erythropolis TA37 strain.

Rhodococcus equi is the etiologic agent of a devastating pneumonia of sporadic incidence in foals. The purpose of this study was to evaluate the influence of R. equi on the superoxide anion production, measured spectrophotometrically as the reduction of cytochrome C, and hexose monophosphate shunt activity, measured by /sup 14/CO/sub 2/ liberation from /sup 14/C-1-D-glucose, of alveolar macrophages from horses. Alveolar macrophages were harvested from 6 anesthetized, healthy, light-breed, adult horses by bronchoalveolar lavage. Following a randomized complete block design, the suspension of cells was divided into aliquots of 10/sup 6/ viable alveolar macrophages which were exposed to 1, 10 or 100 g. of opsonized R. equi or opsonized zymosan A at 37 C for 2 hours. In this study the respiratory burst of equine alveolar macrophages was only evidenced by the hexose monophosphate shunt activity and superoxide anion was not coincidentally produced. Rhodococcus equi did not adversely affect that response. The insignificant superoxide anion production by the alveolar macrophages suggests that this may not be a significant oxygen metabolite in those cells.

Rhodococcus equi is a facultative intracellular, Gram-positive, soilborne actinomycete which can cause severe pyogranulomatous pneumonia with abscessation in young horses (foals) and in immunocompromised people, such as persons with AIDS. All strains of R. equi isolated from foals and approximately a third isolated from humans contain a large, ~81-kb plasmid which is essential for the intramacrophage growth of the organism and for virulence in foals and murine in vivo model systems. We found that the entire virulence plasmid could be transferred from plasmid-containing strains of R. equi (donor) to plasmid-free R. equi strains (recipient) at a high frequency and that plasmid transmission reestablished the capacity for intracellular growth in macrophages. Plasmid transfer required living cells and cell-to-cell contact and was unaffected by the presence of DNase, factors pointing to conjugation as the major means of genetic transfer. Deletion of a putative relaxase-encoding gene, traA, located in the proposed conjugative region of the plasmid, abolished plasmid transfer. Reversion of the traA mutation restored plasmid transmissibility. Finally, plasmid transmission to other Rhodococcus species and some additional related organisms was demonstrated. This is the first study showing a virulence plasmid transfer in R. equi, and it establishes a mechanism by which the virulence plasmid can move among bacteria in the soil.

This study focuses on interactions between aerobic soil-derived hydrocarbon degrading bacteria and a suite of perfluorocarboxylic acids and perfluoroalkylsulfonates that are found in aqueous film-forming foams used for fire suppression. No effect on toluene degradation rate or induction time was observed when active cells of Rhodococcus jostii strain RHA1 were exposed to toluene and a mixture of perfluoroalkyl acids (PFAAs) including perfluorooctanoic acid (PFOA) and perfluorooctanesulfonate (PFOS) at concentrations near the upper bounds of groundwater relevance (11 PFAAs at 10 mg/L each). However, exposure to aqueous PFAA concentrations above 2 mg/L (each) was associated with enhanced aggregation of bacterial cells and significant increases in extracellular polymeric substance production. Flocculation was only observed during exponential growth and not elicited when PFAAs were added to resting incubations; analogous flocculation was also observed in soil enrichments. Aggregation was accompanied by 2- to 3-fold upregulation of stress-associated genes, sigF3 and prmA, during growth of this Rhodococcus in the presence of PFAAs. These results suggest that biological responses, such as microbial stress and biofilm formation, could be more prominent than suppression of co-contaminant biodegradation in subsurface locations where poly- and perfluoroalkyl substances occur with hydrocarbon fuels.

This work has a focus on adaptive capabilities of the actinobacterium Rhodococcus ruber IEGM 326 to cope with drotaverine hydrochloride (DH), a known pharmaceutical pollutant. Cultivation of R. ruber in a nitrogen-limited medium with incubation at the ambient temperature resulted in the formation of cyst-like dormant cells (CLDCs). They maintained viability for 2-7 months, possessed the undetectable respiratory activity and elevated resistance to heating, and had a specific morphology. CLDCs are regarded to ensure long-term survival in various habitats and may be used as storage formulations. R. ruber IEGM 326 was tolerant to DH (MIC, 200 mg/l) and displayed different abilities to degrade this compound, depending on inoculum, temperature, and the presence of glucose as co-oxidized substrate. Thus, the loss of DH (20 mg/l) over 48 h at the optimal temperature (27 ± 2 °C) was 5-8 % in the absence of glucose after inoculating with vegetative cells. The addition of glucose (5 g/l) increased DH degradation up to 46 %. Noteworthy, CLDCs as inoculum were advantageous over vegetative cells to degrade DH at the non-optimal temperature (35 ± 2 °C) at reduced bulk respiratory activity. The obtained results are promising to improve the biodegrading capabilities of other Rhodococcus strains.

Drotaverine [1-(3,4-diethoxybenzylidene)-6,7-diethoxy-1,2,3,4-tetrahydroisoquinoline] hydrochloride, an antispasmodic drug derived from benzylisoquinoline was evaluated for its biodegradability using a bacterial strain Rhodococcus rhodochrous IEGM 608. The experiments were performed under aerobic conditions with rhodococci cultures able to degrade drotaverine. In the presence of glucose, the removal efficiency of drotaverine by free Rhodoccocus cells pre-grown with isoquinoline was above 80 % (200 mg/l, initial concentration) after 25 days. Rhodococcus immobilization on hydrophobized sawdust enhanced the biodegradation process, with the most marked drotaverine loss being observed during the first 5 days of fermentation. High metabolic activity of rhodococcal cells towards drotaverine was confirmed respirometrically. GC-MS analysis of transformation products resulting from drotaverine biodegradation revealed 3,4-diethoxybenzoic acid, 3,4-diethoxybenzaldehyde and 3,4-diethoxybenzoic acid ethyl ester which were detected in the culture medium until drotaverine completely disappeared. Based on these major and other minor metabolites, putative pathways for drotaverine biodegradation were proposed. The obtained data broadened the spectrum of organic xenobiotics oxidized by Rhodoccocus bacteria and proved their potential in decontamination of natural ecosystems from pharma pollutants.

Full Text Available In vitro, a new protocol of plant regeneration in rose was achieved via protocorm-like bodies (PLBs induced from the root-like organs named rhizoids that developed from leaf explants. The development of rhizoids is a critical stage for efficient regeneration, which is triggered by exogenous auxin. However, the role of cytokinin in the control of organogenesis in rose is as yet uncharacterized. The aim of this study was to elucidate the molecular mechanism of cytokinin-modulated rhizoid formation in Rosa canina. Here, we found that cytokinin is a key regulator in the formation of rhizoids. Treatment with cytokinin reduced callus activity and significantly inhibited rhizoid formation in Rosa canina. We further isolated the full-length cDNA of a type-A response regulator gene of cytokinin signaling, RcRR1, from which the deduced amino acid sequence contained the conserved DDK motif. Gene expression analysis revealed that RcRR1 was differentially expressed during rhizoid formation and its expression level was rapidly up-regulated by cytokinin. In addition, the functionality of RcRR1 was tested in Arabidopsis. RcRR1 was found to be localized to the nucleus in GFP-RcRR1 transgenic plants and overexpression of RcRR1 resulted in increased primary root length and lateral root density. More importantly, RcRR1 overexpression transgenic plants also showed reduced sensitivity to cytokinin during root growth; auxin distribution and the expression of auxin efflux carriers PIN genes were altered in RcRR1 overexpression plants. Taken together, these results demonstrate that RcRR1 is a functional type-A response regulator which is involved in cytokinin-regulated rhizoid formation in Rosa canina.

One of the classical functions of the plant hormone cytokinin is the regulation of plastid development, but the underlying molecular mechanisms remain elusive. In this study, we employed a genetic approach to evaluate the role of cytokinin and its signaling pathway in the light-induced development of chloroplasts from etioplasts in Arabidopsis (Arabidopsis thaliana). Cytokinin increases the rate of greening and stimulates ultrastructural changes characteristic for the etioplast-to-chloroplast transition. The steady-state levels of metabolites of the tetrapyrrole biosynthesis pathway leading to the production of chlorophyll are enhanced by cytokinin. This effect of cytokinin on metabolite levels arises due to the modulation of expression for chlorophyll biosynthesis genes such as HEMA1, GUN4, GUN5, and CHLM. Increased expression of HEMA1 is reflected in an enhanced level of the encoded glutamyl-tRNA reductase, which catalyzes one of the rate-limiting steps of chlorophyll biosynthesis. Mutant analysis indicates that the cytokinin receptors ARABIDOPSIS HIS KINASE2 (AHK2) and AHK3 play a central role in this process. Furthermore, the B-type ARABIDOPSIS RESPONSE REGULATOR1 (ARR1), ARR10, and ARR12 play an important role in mediating the transcriptional output during etioplast-chloroplast transition. B-type ARRs bind to the promotors of HEMA1 and LHCB6 genes, indicating that cytokinin-dependent transcription factors directly regulate genes of chlorophyll biosynthesis and the light harvesting complex. Together, these results demonstrate an important role for the cytokinin signaling pathway in chloroplast development, with the direct transcriptional regulation of chlorophyll biosynthesis genes as a key aspect for this hormonal control. PMID:27388681

Cytokinins (CKs) regulate plant development and growth via a two-component signaling pathway. By forward genetic screening, we isolated an Arabidopsis mutant named grow fast on cytokinins 1 (gfc1), whose seedlings grew larger aerial parts on MS medium with CK. gfc1 is allelic to a previously reported cutin mutant defective in cuticular ridges (dcr). GFC1/DCR encodes a soluble BAHD acyltransferase (a name based on the first four enzymes characterized in this family: Benzylalcohol O-acetyltransferase, Anthocyanin O-hydroxycinnamoyltransferase, anthranilate N-hydroxycinnamoyl/benzoyltransferase and Deacetylvindoline 4-O-acetyltransferase) with diacylglycerol acyltransferase (DGAT) activity in vitro and is necessary for normal cuticle formation on epidermis in vivo. Here we show that gfc1 was a CK-insensitive mutant, as revealed by its low regeneration frequency in vitro and resistance to CK in adventitious root formation and dark-grown hypocotyl inhibition assays. In addition, gfc1 had de-etiolated phenotypes in darkness and was therefore defective in skotomorphogenesis. The background expression levels of most type-A Arabidopsis Response Regulator (ARR) genes were higher in the gfc1 mutant. The gfc1-associated phenotypes were also observed in the cutin-deficient glycerol-3-phosphate acyltransferase 4/8 (gpat4/8) double mutant [defective in glycerol-3-phosphate (G3P) acyltransferase enzymes GPAT4 and GPAT8, which redundantly catalyze the acylation of G3P by hydroxyl fatty acid (OH-FA)], but not in the cutin-deficient mutant cytochrome p450, family 86, subfamily A, polypeptide 2/aberrant induction of type three 1 (cyp86A2/att1), which affects the biosynthesis of some OH-FAs. Our results indicate that some acyltransferases associated with cutin formation are involved in CK responses and skotomorphogenesis in Arabidopsis.

Objective: To develop an improved protocol for micropropagation of ethnomedicinally important Scoparia dulcis (S. dulcis) L. Methods: Explants were inoculated on MS basal medium supplemented with kinetin and 6-benzylaminopurine for shoot bud induction. To enhance the shoot induction, various auxins like 3-indoleacetic acid or 3-indolebutyric acid or α-naphthylacetic acid were tested along with 2.32 M KI and 4.44 μM BAP. The regenerated shoots were rooted in half strength MS medium supplemented with various concentrations of IAA, IBA or NAA. After roots were developed, the plantlets were transplanted to pots filled with vermiculate and sand and kept in growth chamber with 70%-80% humidity under 16 h photoperiod. After acclimatization, the plantlets were transferred to the garden and survival percentage was calculated. Data were statistically analyzed and means were compared using Duncan's multiple range test (P<0.05). Results: An in vitro method was developed to induce high frequency shoots regeneration from stem, mature leaf and young leaf explants of S. dulcis. Shoot induction on young leaf explants was most successful in MS medium supplemented with combination of two cytokinins (2.32 μM KI and 4.44 μM BAP) 2.85 μM IAA, 10% CM and 1 483.79 μM adenine sulfate. A single young leaf explant was capable of producing 59 shoots after 13 days of culture. Flower was induced in medium supplemented with combination of KI and BAP. Conclusions:Cytokinins are the key factor to induce the direct shoot regeneration and flowering of S. dulcis.

Full Text Available Cytokinins (CKs regulate plant development and growth via a two-component signaling pathway. By forward genetic screening, we isolated an Arabidopsis mutant named grow fast on cytokinins 1 (gfc1, whose seedlings grew larger aerial parts on MS medium with CK. gfc1 is allelic to a previously reported cutin mutant defective in cuticular ridges (dcr. GFC1/DCR encodes a soluble BAHD acyltransferase (a name based on the first four enzymes characterized in this family: Benzylalcohol O-acetyltransferase, Anthocyanin O-hydroxycinnamoyltransferase, anthranilate N-hydroxycinnamoyl/benzoyltransferase and Deacetylvindoline 4-O-acetyltransferase with diacylglycerol acyltransferase (DGAT activity in vitro and is necessary for normal cuticle formation on epidermis in vivo. Here we show that gfc1 was a CK-insensitive mutant, as revealed by its low regeneration frequency in vitro and resistance to CK in adventitious root formation and dark-grown hypocotyl inhibition assays. In addition, gfc1 had de-etiolated phenotypes in darkness and was therefore defective in skotomorphogenesis. The background expression levels of most type-A Arabidopsis Response Regulator (ARR genes were higher in the gfc1 mutant. The gfc1-associated phenotypes were also observed in the cutin-deficient glycerol-3-phosphate acyltransferase 4/8 (gpat4/8 double mutant [defective in glycerol-3-phosphate (G3P acyltransferase enzymes GPAT4 and GPAT8, which redundantly catalyze the acylation of G3P by hydroxyl fatty acid (OH-FA], but not in the cutin-deficient mutant cytochrome p450, family 86, subfamily A, polypeptide 2/aberrant induction of type three 1 (cyp86A2/att1, which affects the biosynthesis of some OH-FAs. Our results indicate that some acyltransferases associated with cutin formation are involved in CK responses and skotomorphogenesis in Arabidopsis.

Cytokinin, auxin and gibberellin contents in resting and wound-responding potato tubers have not been fully determined and coordinated with wound-healing processes. Using a well-defined wound-healing model system, hormone content and expression of genes associated with hormone turnover were determined in tubers following wounding. Changes in hormone content were coordinated with: (I) formation and completion of the wound closing layer (0-5/6 days), and (II) initiation of phellogen and wound periderm formation (∼ 7 days). Quantifiable amounts of biologically active cytokinins (Z, DZ and IP) were not detected in resting or wound-responding tubers. However, the precursor IPA and catabolic product c-ZOG were found in small amounts in resting and wound-responding tubers. Wound-induced activation of cytokinin biosynthesis was suggested by an increase in t-ZR and c-ZR content at 0.5 days and large increases in IPA and c-ZR content by 3 days and throughout 7 days after wounding suggesting roles in II, but little or no role in I. Expression of key genes involved in cytokinin metabolism followed similar profiles with transcripts decreasing through 3 days and then increasing at 5-7 days after wounding. Both free IAA and IAA-Asp were present in resting tubers. While IAA-Asp was no longer present by 3 days after wounding, IAA content nearly doubled by 5 days and was more than 4-fold greater at 7 days compared to that in resting tuber (0 day) suggesting roles in II, but little or no role in I. Gibberellins were not present in quantifiable amounts in resting or wound-responding tubers. These results suggest that bio-active cytokinins are wound-induced, but their residency is temporal and highly regulated. The transient presence of active cytokinins and corresponding increases in IAA content strongly suggest their involvement in the regulation of wound periderm development. The absence of gibberellins indicates that they are not a regulatory component of wound-healing processes.

• A plant-microbe dual biological system was set up involving the model legume Medicago truncatula and two bacteria, the soil-borne root pathogen Ralstonia solanacearum and the beneficial symbiont Sinorhizobium meliloti. • Comparison of transcriptomes under symbiotic and pathogenic conditions highlighted the transcription factor MtEFD (Ethylene response Factor required for nodule Differentiation) as being upregulated in both interactions, together with a set of cytokinin-related transcripts involved in metabolism, signaling and response. MtRR4 (Response Regulator), a cytokinin primary response gene negatively regulating cytokinin signaling and known as a target of MtEFD in nodulation processes, was retrieved in this set of transcripts. • Refined studies of MtEFD and MtRR4 expression during M. truncatula and R. solanacearum interaction indicated differential kinetics of induction and requirement of central regulators of bacterial pathogenicity, HrpG and HrpB. Similar to MtRR4, MtEFD upregulation during the pathogenic interaction was dependent on cytokinin perception mediated by the MtCRE1 (Cytokinin REsponse 1) receptor. • The use of M. truncatula efd-1 and cre1-1 mutants evidenced MtEFD and cytokinin perception as positive factors for bacterial wilt development. These factors therefore play an important role in both root nodulation and root disease development.

Six-year-old cloned Betula pubescens Ehrh. trees, grown outdoors at 65 degrees 01' N, were cut on six dates during the growing season to study coppice shoot development in relation to root-produced cytokinin-like compounds. Bleeding sap was collected over timed intervals for two days after cutting, and endogenous cytokinin-like compounds were measured by ELISA assay in HPLC-purified fractions of xylem sap. Initiation and development of coppice shoots on the clonally propagated plants were comparable to those in seedlings. Coppice shoot initiation was affected by the time of cutting, diminishing significantly after June. Of the cytokinin-like compounds detected in the xylem sap, zeatin riboside-like (ZR) compounds were present in the highest concentrations, and the concentrations of dihydrozeatin riboside-like (DHZR) and isopentenyladenoside-like (IPA) compounds were approximately one third and one eighth of the ZR concentrations, respectively. The concentration of cytokinin-like compounds was positively correlated with xylem sap flow rate. The export of cytokinin-like compounds, especially DHZR- and ZR-types, was positively correlated with the initiation and elongation rate of coppice shoots, the number of lateral branches, and the radial growth of the more slowly growing coppice shoots. The export of cytokinin-like compounds collected immediately after cutting may represent the basal value for each tree. This value is probably affected by the size and activity of the root system and may be a relevant estimate for predicting the success of coppicing.

3-Ketosteroid dehydrogenases are flavoproteins which play key roles in steroid ring degradation. The enzymes are abundantly present in actinobacteria, including the catabolic powerhouse Rhodococcus jostii and the pathogenic species R. equi and Mycobacterium tuberculosis. The gene for 3-ketosteroid

The permafrost soils of the high elevation McMurdo Dry Valleys are the most cold, desiccating and oligotrophic on Earth. Rhodococcus sp. JG3 is one of very few bacterial isolates from Antarctic Dry Valley permafrost, and displays subzero growth down to -5°C. To understand how Rhodococcus sp. JG3 is able to survive extreme permafrost conditions and be metabolically active at subzero temperatures, we sequenced its genome and compared it to the genomes of 14 mesophilic rhodococci. Rhodococcus sp. JG3 possessed a higher copy number of genes for general stress response, UV protection and protection from cold shock, osmotic stress and oxidative stress. We characterized genome wide molecular adaptations to cold, and identified genes that had amino acid compositions favourable for increased flexibility and functionality at low temperatures. Rhodococcus sp. JG3 possesses multiple complimentary strategies which may enable its survival in some of the harshest permafrost on Earth.

The effect of supplementing either meta-topolin (mT) or N(6)-benzyladenine (BA) requiring cultures with roscovitine (6-benzylamino-2-[1(R)-(hydroxymethyl)propyl]amino-9-isopropylpurine), a cyclin-dependent kinase (CDK) and N-glucosylation inhibitor, and INCYDE (2-chloro-6-(3-methoxyphenyl)aminopurine), an inhibitor of cytokinin (CK) degradation, on the endogenous CK profiles and physiology of banana in vitro was investigated. Growth parameters including multiplication rate and biomass were recorded after 42 days. Endogenous CK levels were quantified using UPLC-MS/MS while the photosynthetic pigment and phenolic contents were evaluated spectrophotometrically. The highest regeneration rate (93 %) was observed in BA + roscovitine while mT + INCYDE plantlets produced most shoots. Treatment with BA + roscovitine had the highest shoot length and biomass. Although not significant, there was a higher proanthocyanidin level in BA + roscovitine treatments compared to the control (BA). The levels of total phenolics and flavonoids were significantly higher in mT + roscovitine treatment than in the mT-treated regenerants. The presence of roscovitine and/or INCYDE had no significant effect on the photosynthetic pigments of the banana plantlets. Forty-seven aromatic and isoprenoid CKs categorized into nine CK-types were detected at varying concentrations. The presence of mT + roscovitine and/or INCYDE increased the levels of O-glucosides while 9-glucosides were higher in the presence of BA. Generally, the underground parts had higher CK levels than the aerial parts; however, the presence of INCYDE increased the level of CK quantified in the aerial parts. From a practical perspective, the use of roscovitine and INCYDE in micropropagation could be crucial in the alleviation of commonly observed in vitro-induced physiological abnormalities.

Responses to drought, heat, and combined stress were compared in tobacco (Nicotiana tabacum L.) plants ectopically expressing the cytokinin oxidase/dehydrogenase CKX1 gene of Arabidopsis thaliana L. under the control of either the predominantly root-expressed WRKY6 promoter or the constitutive 35S promoter, and in the wild type. WRKY6:CKX1 plants exhibited high CKX activity in the roots under control conditions. Under stress, the activity of the WRKY6 promoter was down-regulated and the concomitantly reduced cytokinin degradation coincided with raised bioactive cytokinin levels during the early phase of the stress response, which might contribute to enhanced stress tolerance of this genotype. Constitutive expression of CKX1 resulted in an enlarged root system, a stunted, dwarf shoot phenotype, and a low basal level of expression of the dehydration marker gene ERD10B. The high drought tolerance of this genotype was associated with a relatively moderate drop in leaf water potential and a significant decrease in leaf osmotic potential. Basal expression of the proline biosynthetic gene P5CSA was raised. Both wild-type and WRKY6:CKX1 plants responded to heat stress by transient elevation of stomatal conductance, which correlated with an enhanced abscisic acid catabolism. 35S:CKX1 transgenic plants exhibited a small and delayed stomatal response. Nevertheless, they maintained a lower leaf temperature than the other genotypes. Heat shock applied to drought-stressed plants exaggerated the negative stress effects, probably due to the additional water loss caused by a transient stimulation of transpiration. The results indicate that modulation of cytokinin levels may positively affect plant responses to abiotic stress through a variety of physiological mechanisms.

A method of simultaneous species-specific PCR detection and viability testing of poly(vinyl alcohol) cryogel-entrapped Rhodococcus spp. was developed that allowed the estimation of immobilized Rhodococcus opacus and Rhodococcus ruber survival after their exposure to petroleum hydrocarbon mixture. Spectrophotometric INT assay revealed high tolerance of gel-immobilized rhodococci to petroleum hydrocarbons, while among two Rhodococcus strains studied, R. ruber tolerated better to hydrocarbons compared to R. opacus. These findings were confirmed by respirometry results that showed increased respiratory activity of gel-immobilized Rhodococcus strains after 10-day incubation with 3% (v/v) petroleum hydrocarbon mixture. Moreover, jointly incubated rhodococcal strains demonstrated higher oxidative activities toward petroleum hydrocarbons than individual strains. Both Rhodococcus species were recovered successfully in cryogel granules using 16S rDNA-targeted PCR, even though the granules were previously stained with INT and extracted with ethanol. The method developed can be used for rapid detection and monitoring of gel-immobilized bacterial inocula in bioreactors or contaminated soil systems.

The limitations in current understanding of the molecular mechanisms underlying fruit response to the application of plant growth regulators have increasingly become major challenges in improvement of crop quality. This study aimed to evaluate the response of strawberry to the preharvest application of exogenous cytokinin known as forchlorfenuron (CPPU). Postharvest internal and physiological quality attributes were characterized following storage under different conditions. Hierarchical clustering analysis via a label-free proteomic quantitative approach identified a total of 124 proteins in strawberries across all treatments. The expression profiles of both proteins and genes spanned the ranged role of cytokinin involved in primary and secondary metabolism, stress response, and so on. Eighty-eight proteins and fifty-six proteins were significantly regulated immediately at harvest and after storage, respectively. In general, the glycolysis in strawberry was only regulated by CPPU before storage; in addition to the accelerated photosynthesis and acid metabolism, CPPU application maintained higher capacity of resistance in strawberry to stress stimuli after storage, in comparison to control. Nevertheless, the volatile biosynthesis in strawberry has been suppressed by exogenous CPPU. Novel cytokinin response proteins and processes were identified in addition to the main transcriptomic expression to gain insights into the phytohormone control of fruit postharvest quality. PMID:27250251

Cytokinin is a critical growth regulator for various aspects of plant growth and development. In Arabidopsis, cyto-kinin signaling is mediated by a two-component system-based phosphorelay that transmits a signal from the recep-tors, through histidine phosphotransfer proteins, to the downstream response regulators (ARRs). Of these ARRs, type-A ARR genes, whose transcription can be rapidly induced by cytokinin, act as negative regulators of cytokinin signaling. However, because of functional redundancy, the function of type-A ARR genes in plant growth and de-velopment is not well understood by analyzing loss-of-function mutants. In this study, we performed a comparative functional study on all ten type-A ARR genes by analyzing transgenic plants overexpressing these ARR genes fused to a MYC epitope tag. Overexpression of ARR genes results in a variety of cytokinin-associated phenotypes. Notably, overexpression of different ARR transgenes causes diverse phenotypes, even between phylogenetically closely-related gene pairs, such as within the ARR3-ARR4 and ARRS-ARR6 pairs. We found that the accumulation of a subset of ARR proteins (ARR3, ARR5, ARR7, ARR16 and ARR17; possibly ARR8 and ARR15) is increased by MGI32, a spe-cific proteasomal inhibitor, indicating that stability of these proteins is regulated by proteasomal degradation. More-over, similar to that of previously characterized ARR5, ARR6 and ARR7, stability of ARR16 and ARR17, possibly including ARR8 and ARRI5, is regulated by cytokinin. These results suggest that type-A ARR proteins are regulated by a combinatorial mechanism involving both the cytokinin and proteasome pathways, thereby executing distinctive functions in plant growth and development.

In this study, biodesulfurization (BDS) was carried out using immobilized Rhodococcus erythropolis KA2-5-1 in n-tetradecane containing dibenzothiophene (DBT) as a model oil (n-tetradecane/immobilized cell biphasic system). The cells were immobilized by entrapping them with calcium alginate, agar, photo-crosslinkable resin prepolymers (ENT-4000 and ENTP-4000), and urethane prepolymers (PU-3 and PU-6); and it was found that ENT-4000-immobilized cells had the highest DBT desulfurization activity in the model oil system without leakage of cells from the support. Furthermore, ENT-4000-immobilized cells could catalyze BDS repeatedly in this system for more than 900 h with reactivation; and recovery of both the biocatalyst and the desulfurized model oil was easy. This study would give a solution to the problems in BDS, such as the troublesome process of recovering desulfurized oil and the short life of BDS biocatalysts. (orig.)

Rhodococcus erythropolis strain UPV-1 is able to grow on phenol as the only carbon and energy source and to remove formaldehyde completely from both synthetic and industrial wastewater. The rate of formaldehyde removal is independent of either initial biomass or formaldehyde concentration. The presence of viable, intact cells is strictly necessary for this removal to take place. Discontinuous and continuous formaldehyde-feed systems were successfully tested with synthetic wastewater in shaken flasks. Once biodegradation was well established in model synthetic wastewater, a real wastewater sample was obtained from a local phenolic and melamine resin-manufacturing company. Incubation of biomass with this wastewater at subtoxic concentrations of formaldehyde resulted in the complete removal of the pollutant. Parameters, such as chemical oxygen demand and toxicity, were assessed as indicators of wastewater cleanup progress.

The bacterial strain J3 was isolated from soil by selective enrichment on mineral medium containing 4-nitrocatechol as the sole carbon and energy source. This strain was identified as Rhodococcus wratislaviensis on the basis of morphology, biochemical, physiological and chemotaxonomic characterization and complete sequencing of the 16S rDNA gene. The isolated bacterium could utilize 4-nitrocatechol, 3-nitrophenol and 5-nitroguaiacol as sole carbon and energy sources. Stoichiometric release of nitrites was measured during degradation of 4-nitrocatechol both in growing cultures and for stationary phase cells. The J3 strain was unable to degrade 4-nitroguaiacol, 2-nitrophenol, 4-nitrophenol, 2,4-dinitrobenzoic acid, 4,5-dimethoxy-2-nitrobenzoic acid and 2,3-difluoro-6-nitrophenol. The J3 strain is deposited in the Czech Collection of Microorganisms as CCM 4930.

Full Text Available Rhodococcus equi is a facultative intracellular pathogen of macrophages and the causative agent of foal pneumonia. R. equi virulence is usually assessed by analyzing intracellular growth in macrophages by enumeration of bacteria following cell lysis, which is time consuming and does not allow for a high throughput analysis. This paper describes the use of an impedance based real-time method to characterize proliferation of R. equi in macrophages, using virulent and attenuated strains lacking the vapA gene or virulence plasmid. Image analysis suggested that the time-dependent cell response profile (TCRP is governed by cell size and roundness as well as cytoxicity of infecting R. equi strains. The amplitude and inflection point of the resulting TCRP were dependent on the multiplicity of infection as well as virulence of the infecting strain, thus distinguishing between virulent and attenuated strains.

A 2-month-old foal with septic shock and severe respiratory distress was referred to the Veterinary Teaching Hospital. Due to poor prognosis, the foal was euthanized. Histopathology showed lesions suggestive of Rhodococcus equi infection associated with a diffuse interstitial infiltrate of foamy macrophages and syncytial cells presenting large acidophilic intranuclear inclusion bodies, fibrin exudates and hyaline membranes. Bacteriological examination from lung and respiratory exudates confirmed R. equi infection, whilst immunohistochemistry and PCR yielded a positive result for Equid herpesvirus type 1 (EHV-1). Several etiologies have been proposed for bronchointerstitial pneumonia in foals, although a multifactorial origin for this lesional pattern could be possible. This work is the first one describing a combined EHV-1 and R. equi infection in a foal affected with bronchointerstitial pneumonia.

Full Text Available Rhodococcus equi infection treatment is usually a macrolide (azithromycin - AZM, clarithromycin - CLR and erythromycin - ERY and rifampicin combination. However, resistance cases have been reported, especially for ERY. In view of the need of a study about Brazilian isolates susceptibility profile, this study aimed to characterize the minimum inhibitory concentration (MIC of the macrolides - AZM, CLR and ERY - against 44 R. equi isolates. It was found all isolates CLR and AZM sensitive; however, for ERY, 27% (12/44 were classified as intermediate sensitivity. R. equi Brazilian isolates used here showed a large susceptibility profile, except against ERY, for which it was observed some resistance evidence. In order to avoid failures in the equine rhodococcosis therapy it was highlighted the importance of microbiological culture and antimicrobial susceptibility testing in vitro before beginning treatment

Rhodococcus equi is a facultative intracellular pathogen of macrophages and the causative agent of foal pneumonia. R. equi virulence is usually assessed by analyzing intracellular growth in macrophages by enumeration of bacteria following cell lysis, which is time consuming and does not allow for a high throughput analysis. This paper describes the use of an impedance based real-time method to characterize proliferation of R. equi in macrophages, using virulent and attenuated strains lacking the vapA gene or virulence plasmid. Image analysis suggested that the time-dependent cell response profile (TCRP) is governed by cell size and roundness as well as cytoxicity of infecting R. equi strains. The amplitude and inflection point of the resulting TCRP were dependent on the multiplicity of infection as well as virulence of the infecting strain, thus distinguishing between virulent and attenuated strains.

Full Text Available The influence of different nutrients on biosurfactant production by Rhodococcus erythropolis was investigated. Increasing the concentration of phosphate buffer from 30 up through 150 mmol/L stimulated an increase in biosurfactant production, which reached a maximum concentration of 285 mg/L in shaken flasks. Statistical analysis showed that glycerol, NaNO3,MgSO4 and yeast extract had significant effects on production. The results were confirmed in a batchwise bioreactor, and semi-growth-associated production was detected. Reduction in the surface tension, which indicates the presence of biosurfactant, reached a value of 38 mN/m at the end of 35 hours. Use of the produced biosurfactant for washing crude oil-contaminated soil showed that 2 and 4 times the critical micellar concentration (CMC were able to remove 97 and 99% of the oil, respectively, after 1 month of impregnation.

Rhodococcus rhodochrous NCIMB 11216 produced nitrile hydratase (320 nkat mg of protein−1) and amidase activity (38.4 nkat mg of protein−1) when grown on a medium containing propionitrile. These enzymes were able to hydrolyze nitrile groups of both granular polyacrylonitriles (PAN) and acrylic fibers. Nitrile groups of PAN40 (molecular mass, 40 kDa) and PAN190 (molecular mass, 190 kDa) were converted into the corresponding carbonic acids to 1.8 and 1.0%, respectively. In contrast, surfacial nitrile groups of acrylic fibers were only converted to the corresponding amides. X-ray photoelectron spectroscopy analysis showed that 16% of the surfacial nitrile groups were hydrolyzed by the R. rhodochrous enzymes. Due to the enzymatic modification, the acrylic fibers became more hydrophilic and thus, adsorption of dyes was enhanced. This was indicated by a 15% increase in the staining level (K/S value) for C.I. Basic Blue 9. PMID:10742253

Full Text Available We report the genome of the facultative intracellular parasite Rhodococcus equi, the only animal pathogen within the biotechnologically important actinobacterial genus Rhodococcus. The 5.0-Mb R. equi 103S genome is significantly smaller than those of environmental rhodococci. This is due to genome expansion in nonpathogenic species, via a linear gain of paralogous genes and an accelerated genetic flux, rather than reductive evolution in R. equi. The 103S genome lacks the extensive catabolic and secondary metabolic complement of environmental rhodococci, and it displays unique adaptations for host colonization and competition in the short-chain fatty acid-rich intestine and manure of herbivores--two main R. equi reservoirs. Except for a few horizontally acquired (HGT pathogenicity loci, including a cytoadhesive pilus determinant (rpl and the virulence plasmid vap pathogenicity island (PAI required for intramacrophage survival, most of the potential virulence-associated genes identified in R. equi are conserved in environmental rhodococci or have homologs in nonpathogenic Actinobacteria. This suggests a mechanism of virulence evolution based on the cooption of existing core actinobacterial traits, triggered by key host niche-adaptive HGT events. We tested this hypothesis by investigating R. equi virulence plasmid-chromosome crosstalk, by global transcription profiling and expression network analysis. Two chromosomal genes conserved in environmental rhodococci, encoding putative chorismate mutase and anthranilate synthase enzymes involved in aromatic amino acid biosynthesis, were strongly coregulated with vap PAI virulence genes and required for optimal proliferation in macrophages. The regulatory integration of chromosomal metabolic genes under the control of the HGT-acquired plasmid PAI is thus an important element in the cooptive virulence of R. equi.

The cell-free extract of locally isolated Rhodococcus UKMP-5M strain was used as an alternative to develop greener and cost effective cyanide removal technology. The present study aims to assess the viability of the cell-free extract to detoxify high concentrations of cyanide which is measured through the monitoring of protein concentration and specific cyanide-degrading activity. When cyanide-grown cells were subjected to grinding in liquid nitrogen which is relatively an inexpressive and fast cell disruption method, highest cyanide-degrading activity of 0.63 mM min(-1) mg(-1) protein was obtained in comparison to enzymatic lysis and agitation with fine glass beads. The cell-free extracts managed to degrade 80% of 20 mM KCN within 80 min and the rate of cyanide consumption increased linearly as the concentration of protein was raised. In both cases, the addition of co-factor was not required which proved to be advantageous economically. The successful formation of ammonia and formate as endproducts indicated that the degradation of cyanide by Rhodococcus UKMP-5M proceeded via the activity of cyanidase and the resulting non-toxic products are safe for disposal into the environment. Further verification with SDS-PAGE revealed that the molecular weight of the active enzyme was estimated to be 38 kDa, which is consistent with previously reported cyanidases. Thus, the utilization of cell-free extracts as an alternative to live microbial in cyanide degradation offers numerous advantageous such as the potential to tolerate and degrade higher concentration of cyanide and total reduction in the overall cost of operation since the requirement for nutrient support is irrelevant.

We report the genome of the facultative intracellular parasite Rhodococcus equi, the only animal pathogen within the biotechnologically important actinobacterial genus Rhodococcus. The 5.0-Mb R. equi 103S genome is significantly smaller than those of environmental rhodococci. This is due to genome expansion in nonpathogenic species, via a linear gain of paralogous genes and an accelerated genetic flux, rather than reductive evolution in R. equi. The 103S genome lacks the extensive catabolic and secondary metabolic complement of environmental rhodococci, and it displays unique adaptations for host colonization and competition in the short-chain fatty acid–rich intestine and manure of herbivores—two main R. equi reservoirs. Except for a few horizontally acquired (HGT) pathogenicity loci, including a cytoadhesive pilus determinant (rpl) and the virulence plasmid vap pathogenicity island (PAI) required for intramacrophage survival, most of the potential virulence-associated genes identified in R. equi are conserved in environmental rhodococci or have homologs in nonpathogenic Actinobacteria. This suggests a mechanism of virulence evolution based on the cooption of existing core actinobacterial traits, triggered by key host niche–adaptive HGT events. We tested this hypothesis by investigating R. equi virulence plasmid-chromosome crosstalk, by global transcription profiling and expression network analysis. Two chromosomal genes conserved in environmental rhodococci, encoding putative chorismate mutase and anthranilate synthase enzymes involved in aromatic amino acid biosynthesis, were strongly coregulated with vap PAI virulence genes and required for optimal proliferation in macrophages. The regulatory integration of chromosomal metabolic genes under the control of the HGT–acquired plasmid PAI is thus an important element in the cooptive virulence of R. equi. PMID:20941392

VapD is one of a set of highly homologous virulence-associated proteins from the multi-host pathogen Rhodococcus equi. The crystal structure reveals an eight-stranded β-barrel with a novel fold and a glycine rich ‘bald’ surface. Rhodococcus equi is a multi-host pathogen that infects a range of animals as well as immune-compromised humans. Equine and porcine isolates harbour a virulence plasmid encoding a homologous family of virulence-associated proteins associated with the capacity of R. equi to divert the normal processes of endosomal maturation, enabling bacterial survival and proliferation in alveolar macrophages. To provide a basis for probing the function of the Vap proteins in virulence, the crystal structure of VapD was determined. VapD is a monomer as determined by multi-angle laser light scattering. The structure reveals an elliptical, compact eight-stranded β-barrel with a novel strand topology and pseudo-twofold symmetry, suggesting evolution from an ancestral dimer. Surface-associated octyl-β-d-glucoside molecules may provide clues to function. Circular-dichroism spectroscopic analysis suggests that the β-barrel structure is preceded by a natively disordered region at the N-terminus. Sequence comparisons indicate that the core folds of the other plasmid-encoded virulence-associated proteins from R. equi strains are similar to that of VapD. It is further shown that sequences encoding putative R. equi Vap-like proteins occur in diverse bacterial species. Finally, the functional implications of the structure are discussed in the light of the unique structural features of VapD and its partial structural similarity to other β-barrel proteins.

Full Text Available We report the genome of the facultative intracellular parasite Rhodococcus equi, the only animal pathogen within the biotechnologically important actinobacterial genus Rhodococcus. The 5.0-Mb R. equi 103S genome is significantly smaller than those of environmental rhodococci. This is due to genome expansion in nonpathogenic species, via a linear gain of paralogous genes and an accelerated genetic flux, rather than reductive evolution in R. equi. The 103S genome lacks the extensive catabolic and secondary metabolic complement of environmental rhodococci, and it displays unique adaptations for host colonization and competition in the short-chain fatty acid-rich intestine and manure of herbivores--two main R. equi reservoirs. Except for a few horizontally acquired (HGT pathogenicity loci, including a cytoadhesive pilus determinant (rpl and the virulence plasmid vap pathogenicity island (PAI required for intramacrophage survival, most of the potential virulence-associated genes identified in R. equi are conserved in environmental rhodococci or have homologs in nonpathogenic Actinobacteria. This suggests a mechanism of virulence evolution based on the cooption of existing core actinobacterial traits, triggered by key host niche-adaptive HGT events. We tested this hypothesis by investigating R. equi virulence plasmid-chromosome crosstalk, by global transcription profiling and expression network analysis. Two chromosomal genes conserved in environmental rhodococci, encoding putative chorismate mutase and anthranilate synthase enzymes involved in aromatic amino acid biosynthesis, were strongly coregulated with vap PAI virulence genes and required for optimal proliferation in macrophages. The regulatory integration of chromosomal metabolic genes under the control of the HGT-acquired plasmid PAI is thus an important element in the cooptive virulence of R. equi.

We previously showed that the facultative intracellular pathogen Rhodococcus equi produces a nondiffusible and catecholate-containing siderophore (rhequibactin) involved in iron acquisition during saprophytic growth. Here, we provide evidence that the rhbABCDE cluster directs the biosynthesis of a hydroxamate siderophore, rhequichelin, that plays a key role in virulence. The rhbC gene encodes a nonribosomal peptide synthetase that is predicted to produce a tetrapeptide consisting of N(5)-formyl-N(5)-hydroxyornithine, serine, N(5)-hydroxyornithine, and N(5)-acyl-N(5)-hydroxyornithine. The other rhb genes encode putative tailoring enzymes mediating modification of ornithine residues incorporated into the hydroxamate product of RhbC. Transcription of rhbC was upregulated during growth in iron-depleted medium, suggesting that it plays a role in iron acquisition. This was confirmed by deletion of rhbCD, rendering the resulting strain R. equi SID2 unable to grow in the presence of the iron chelator 2,2-dipyridyl. Supernatant of the wild-type strain rescued the phenotype of R. equi SID2. The importance of rhequichelin in virulence was highlighted by the rapid increase in transcription levels of rhbC following infection and the inability of R. equi SID2 to grow within macrophages. Unlike the wild-type strain, R. equi SID2 was unable to replicate in vivo and was rapidly cleared from the lungs of infected mice. Rhequichelin is thus a key virulence-associated factor, although nonpathogenic Rhodococcus species also appear to produce rhequichelin or a structurally closely related compound. Rhequichelin biosynthesis may therefore be considered an example of cooption of a core actinobacterial trait in the evolution of R. equi virulence.

Bacteria of the genus Rhodococcus are well known for their ability to degrade a large range of organic compounds. Some rhodococci are free-living, saprophytic bacteria; others are animal and plant pathogens. Recently, several studies have shown that their genomes encode putative pathways for the synthesis of a large number of specialized metabolites that are likely to be involved in microbe-microbe and host-microbe interactions. To systematically explore the specialized metabolic potential of this genus, we here performed a comprehensive analysis of the biosynthetic coding capacity across publicly available rhododoccal genomes, and compared these with those of several Mycobacterium strains as well as that of their mutual close relative Amycolicicoccus subflavus. Comparative genomic analysis shows that most predicted biosynthetic gene cluster families in these strains are clade-specific and lack any homology with gene clusters encoding the production of known natural products. Interestingly, many of these clusters appear to encode the biosynthesis of lipopeptides, which may play key roles in the diverse environments were rhodococci thrive, by acting as biosurfactants, pathogenicity factors or antimicrobials. We also identified several gene cluster families that are universally shared among all three genera, which therefore may have a more 'primary' role in their physiology. Inactivation of these clusters by mutagenesis might help to generate weaker strains that can be used as live vaccines. The genus Rhodococcus thus provides an interesting target for natural product discovery, in view of its large and mostly uncharacterized biosynthetic repertoire, its relatively fast growth and the availability of effective genetic tools for its genomic modification.

We evaluated the capacity of the plant growth regulator thidiazuron (TDZ), a substituted phenylurea with high cytokinin-like activity, to promote organogenesis in petals and leaves of several carnation cultivars (Dianthus spp.), combined with 1-naphthaleneacetic acid (NAA). The involvement of the endogenous auxin indole-3-acetic acid (IAA) and purine-type cytokinins was also studied. Shoot differentiation was found to depend on the explant, cultivar and balance of growth regulators. TDZ alone (0.5 and 5.0 micromol/L) as well as synergistically with NAA (0.5 and 5.0 micromol/L) promoted shoot organogenesis in petals, and was more active than N6-benzyladenine. In petals of the White Sim cultivar, TDZ induced cell proliferation in a concentration-dependent manner and, on day 7 of culture, the proportion of meristematic regions in those petals allowed the prediction of shoot regeneration capacity after 30 days of culture. Immunolocalization of CK ribosides, N6-(delta2-isopentenyl)adenosine, zeatin riboside (ZR) and dihydrozeatin riboside (DHZR), in organogenic petals showed them to be highly concentrated in the tips of bud primordia and in the regions with proliferation capacity. All of them may play a role in cell proliferation, and possibly in differentiation, during the organogenic process. After seven days of culture of White Sim petals, NAA may account for the changes found in the levels of IAA and DHZR, whereas TDZ may be responsible for the remarkable increases in N6-(delta2-isopentenyl)adenine (iP) and ZR. ZR is induced by low TDZ concentrations (0.0-0.005 micromol/L), whereas iP, that correlates with massive cell proliferation and the onset of shoot differentiation, is associated with high TDZ levels (0.5 micromol/L). In addition to the changes observed in quantification and in situ localization of endogenous phytohormones during TDZ-induced shoot organogenesis, we propose that TDZ also promotes growth directly, through its own biological activity. To our

对Rhodococcus sp.BX2菌降解乙腈的特性及其降解途径进行了研究.结果显示,在底物浓度为800mg·L-1,接种量为1.0%,培养温度为35℃,环境pH为7.5的条件下,16h时Rhodococcus sp.BX2菌对乙腈的降解率为95.98%;添加葡萄糖可在培养初期加快Rhodococcus sp.BX2菌的生长和对乙腈的降解,蔗糖、乙酰胺和尿素对其影响不大.将BX2菌接种到含有高乙腈浓度（25000mg·L-1）的合成废水中,培养180h后,乙腈降解率可达88.59%.在催化反应60min后,Rhodococcus sp.BX2腈水合酶与腈水解酶的总酶活可达到422.81U·mL-1,对其相关基因序列的分析结果表明,Rhodococcus sp.BX2中同时存在腈水解酶基因和腈水合酶基因,因此,确定乙腈的降解主要由腈水合酶途径完成,可能同时存在腈水解酶的降解途径.%The characteristics and pathway of acetonitrile degradation by Rhodococcus sp.BX2 were investigated in this study. Results showed that with the initial acetonitrile concentration of 800 mg · L-1, the degradation rate was 95.98% in 16 hours under the condition of inoculum 1.0%, 35 ℃ and pH value 7.5.Glucose could accelerate the degradation of acetonitrile in the initial period, while sucrose, acetamide and urea had slight impact. The degradation rate could reach 88.59% when BX2 was cultured in the synthetic wastewater with high concentration of acetonitrile （25000 mg · L-1） for 180 hours. Total enzyme activities was 422.81 U · mL-1 when incubated for 60 minutes. The results of related genes sequence showed that Rhodococcus sp BX2 had both nitrile hydrolase gene and nitrile hydratase gene. The degradation pathway of acetonitrile by Rhodococcus sp. BX2 was mainly nitrile hydratase （NHase）, with possible pathway of the nitrile hydrolase.

Full Text Available Soil destruction by abiotic environmental conditions, such as high salinity, has resulted in dramatic losses of arable land, giving rise to the need of studying mechanisms of plant adaptation to salt stress aimed at creating salt-tolerant plants. Recently, it has been reported that cytokinins (CKs regulate plant environmental stress responses through two-component systems. A decrease in endogenous CK levels could enhance salt and drought stress tolerance. Here, we have investigated the global transcriptional change caused by a reduction in endogenous CK content under both normal and salt stress conditions. Ten-day-old Arabidopsis thaliana wild-type (WT and CK-deficient ipt1,3,5,7 plants were transferred to agar plates containing either 0 mM (control or 200 mM NaCl and maintained at normal growth conditions for 24 h. Our experimental design allowed us to compare transcriptome changes under four conditions: WT-200 mM vs. WT-0 mM, ipt1,3,5,7-0 mM vs. WT-0 mM, ipt1,3,5,7-200 mM vs. ipt1,3,5,7-0 mM and ipt1,3,5,7-200 mM vs. WT-200 mM NaCl. Our results indicated that the expression of more than 10% of all of the annotated Arabidopsis genes was altered by CK deficiency under either normal or salt stress conditions when compared to WT. We found that upregulated expression of many genes encoding either regulatory proteins, such as NAC, DREB and ZFHD transcription factors and the calcium sensor SOS3, or functional proteins, such as late embryogenesis-abundant proteins, xyloglucan endo-transglycosylases, glycosyltransferases, glycoside hydrolases, defensins and glyoxalase I family proteins, may contribute to improved salt tolerance of CK-deficient plants. We also demonstrated that the downregulation of photosynthesis-related genes and the upregulation of several NAC genes may cause the altered morphological phenotype of CK-deficient plants. This study highlights the impact of CK regulation on the well-known stress-responsive signaling pathways, which

Inducing expression and the reaction characteristic of nitrile hydratase (NHase) from Rhodococcus sp.SHZ-1 were investigated. The results showed that the expression of NHase was greatly enhanced with the cooperation of acrylonitrile and ammonium chloride as inducer in the medium and the specific activity of NHase was increased of 44%. Then the temperature, pH, concentration of acrylonitrile and acrylamide were evaluated, which affected the activity and reaction characteristic of NHase. It was found that the temperature and concentration of acrylamide were the most important factors for the catalyzation of NHase. The optimal catalysis temperature of NHase from Rhodococcus sp. SHZ-1 was 30℃, and the activation energy of the hydration of NHase was 90.2kJ·mol-1 in the temperature range from 5℃ to 30℃. Km of NHase was 0.095mol.L-1 using acrylonitrile(AN)as substrate, and NHase activity was inhibited seriously when acrylonitrile concentration was up to 40g·L-1, the substrate inhibition constant Ki is 0.283mol·L-1. Moreover, the NHase from Rhodococcus sp. SHZ-1 had very strong tolerance to acrylamide, in which the final concentration of acrylamide reached to 642g·L-1 and the residual activity of NHase still maintained 8.6% of the initial enzyme activity.

Effects of some nitriles and amides, as well as glucose and ammonium, on the growth and the nitrile hydratase (EC 4.2.1.84) activity of the Rhodococcus sp. strain gt1 isolated from soil were studied. The activity of nitrile hydratase mainly depended on carbon and nitrogen supply to cells. The activity of nitrile hydratase was high in the presence of glucose and ammonium at medium concentrations and decreased at concentrations of glucose more than 0.3%. Saturated unsubstituted aliphatic nitriles and amides were found to be a good source of nitrogen and carbon. However, the presence of nitriles and amides in the medium was not absolutely necessary for the expression of the activity of nitrile hydratase isolated from the Rhodococcus sp. strain gt1.

We report here on a comparative developmental profile of plant hormone cytokinins in relation to cell size, cell number and endoreduplication in developing maize caryopsis of a cell wall invertase-deficient miniature1 (mn1) seed mutant and its wild type, Mn1, genotype. Both genotypes showed extremely high levels of total cytokinins during the very early stages of development, followed by a marked and genotype specific reduction. While the decrease of cytokinins in Mn1 was associated with their deactivation by 9-glucosylation, the absolute and the relative part of active cytokinin forms was higher in the mutant. During the exponential growth phase of endosperm between 6 d after pollination and 9 d after pollination, the mean cell doubling time, the absolute growth rate and the level of endoreduplication were similar in the two genotypes. However, the entire duration of growth was longer in Mn1 compared with mn1, resulting in a significantly higher cell number in the Mn1 endosperm. These data correlate with the previously reported peak levels of the Mn1-encoded cell wall invertase-2 (INCW2) at 12 d after pollination in the Mn1 endosperm. A model showing possible crosstalk among cytokinins, cell cycle and cell wall invertase as causal to increased cell number and sink strength of the Mn1 developing endosperm is discussed.

A comprehensive range of cytokinins (CK) was identified and quantified by gas chromatography-mass spectrometry in tissues of and in xylem and phloem serving developing white lupine (Lupinus albus) fruits. Analyses were initiated at anthesis and included stages of podset, embryogenesis, and seed filling up to physiological maturation 77 d post anthesis (DPA). In the first 10 DPA, fertilized ovaries destined to set pods accumulated CK. The proportion of cis-CK:trans-CK isomers was initially 10:1 but declined to less than 1:1. In ovaries destined to abort, the ratio of cis-isomers to trans-isomers remained high. During early podset, accumulation of CK (30–40 pmol ovary−1) was accounted for by xylem and phloem translocation, both containing more than 90% cis-isomers. During embryogenesis and early seed filling (40–46 DPA), translocation accounted for 1% to 14% of the increases of CK in endosperm (20 nmol fruit−1) and seed coat (15 nmol fruit−1), indicating synthesis in situ. High CK concentrations in seeds (0.6 μmol g−1 fresh weight) were transient, declining rapidly to less than 1% of maximum levels by physiological maturity. These data pose new questions about the localization and timing of CK synthesis, the significance of translocation, and the role(s) of CK forms in reproductive development. PMID:10938375

Wild-type (WT) and transgenic tobacco plants overexpressing isopentenyltransferase (IPT), a gene coding the rate-limiting step in cytokinin (CKs) synthesis, were grown under limited nitrogen (N) conditions to evaluate the role of CKs in NUE (N-use efficiency) and in different parameters that determine the quality of tobacco leaves. The results indicate that WT tobacco plants submitted to N deficiency show a decline in the leaf/root ratio, associated with a decrease in the NUE and in tobacco-leaf quality, defined by an increase in the quantity of nicotine. On the contrary, the transgenic plants submitted to N deficiency maintained the leaf/root ratio, presenting a higher NUE and greater quality of tobacco leaves than the WT plants, as the latter showed reduced nicotine and an increase in reducing sugars under severe N-deficiency conditions. Therefore, the overexpression of CKs under N deficiency could be a useful tool to improve tobacco cultivation, given that it could reduce N-fertilizer application and thereby provide economic savings and environmental benefits, maintaining yield and improving tobacco leaf quality.

Full Text Available In nature, annual plants respond to abiotic stresses by activating a specific genetic program leading to early flowering and accelerated senescence. Although, in nature, this phenomenon supports survival under unfavorable environmental conditions, it may have negative agro-economic impacts on crop productivity. Overcoming this genetic programing by cytokinins (CK has recently been shown in transgenic plants that overproduce CK. These transgenic plants displayed a significant increase in plant productivity under drought stress conditions. We investigated the role of CK in reverting the transcriptional program that is activated under abiotic stress conditions and allowing sustainable plant growth. We employed 2 complementary approaches: Ectopic overexpression of CK, and applying exogenous CK to detached Arabidopsis leaves. Transgenic Arabidopsis plants transformed with the isopentyltransferase (IPT gene under the regulation of the senescence associated receptor kinase (SARK promoter displayed a significant drought resistance. A transcriptomic analysis using RNA sequencing was performed to explore the response mechanisms under elevated CK levels during salinity stress. This analysis showed that under such stress, CK triggered transcriptional reprograming that resulted in attenuated stress-dependent inhibition of vegetative growth and delayed premature plant senescence. Our data suggest that elevated CK levels led to stress tolerance by retaining the expression of genes associated with plant growth and metabolism whose expression typically decreases under stress conditions. In conclusion, we hypothesize that CK allows sustainable plant growth under unfavorable environmental conditions by activating gene expression related to growth processes and by preventing the expression of genes related to the activation of premature senescence.

It has been reported that pigmentation in plants is stimulated by light and cytokinin (CTK); however, the signaling pathways and the relationship between light and CTK involved in the regulation of anthocyanin accumulation remain to be elucidated. We investigated (i) the role of blue light (BL) and CTK in anthocyanin accumulation; and (ii) the relationship between BL and CTK in wild type (WT) and hy4 mutants of Arabidopsis thaliana. Two-d-old seedlings grown on medium with or without kinetin (KT) or zeatin (ZT) in darkness were irradiated using BL at different fluence rates for 3 d before the anthocyanin content was determined using a spectrophotometric method. Anthocyanin accumulation was strongly induced by BL in WT seedlings but not in hy4 seedlings, which demonstrated that CRY1 is the main photoreceptor for BL. Both KT and ZT enhanced the response of the WT seedlings to BL in a dose-dependent manner, whereas they were not sufficient to promote anthocyanin accumulation in darkness. In addition, data from experiments using the hy4 mutant showed that the CTK effect of BL was also CRY1-dependent. The results from experiments with three different treatment programs showed that the relationship between BL and KT in anthocyanin accumulation of Arabidopsis seedlings seems neither multiplicative nor additive coaction, but rather interaction. BL is necessary for anthocyanin accumulation, and KT might be involved in the BL signaling pathway.

When inoculated onto maize leaves at the onset of senescence, the hemibiotroph Colletotrichum graminicola causes green islands that are surrounded by senescing tissue. Taking advantage of green islands as indicators of sites of the establishment of successful infection and of advanced high-performance liquid chromatography tandem mass spectrometry methodology, we analyzed changes in the patterns and levels of cytokinins (CK) at high spatial and analytical resolution. Twenty individual CK were detected in green islands. Levels of cis-zeatin-9-riboside and cis-zeatin-9-riboside-5'-monophosphate increased drastically, whereas that of the most prominent CK, cis-zeatin-O-glucoside, decreased. The fungus likely performed these conversions because corresponding activities were also detected in in vitro cultures amended with CK. We found no evidence that C. graminicola is able to synthesize CK entirely de novo in minimal medium but, after adding dimethylallyl diphosphate, a precursor of CK biosynthesis occurring in plants, a series of trans-zeatin isoforms (i.e., trans-zeatin-9-riboside-5'-monophosphate, trans-zeatin-9-riboside, and trans-zeatin) was formed. After applying CK onto uninfected leaves, transcripts of marker genes for senescence, photosynthesis, and assimilate distribution were measured by quantitative reverse-transcribed polymerase chain reaction; furthermore, pulse-amplitude modulation chlorophyll fluorometry and single-photon avalanche diode analyses were conducted. These experiments suggested that modulation of CK metabolism at the infection site affects host physiology.

A wheat stay-green mutant, tasg1, was observed to exhibit significantly delayed senescence in the late developmental stage. The photosynthetic capacity of the flag leaf was greater in tasg1 than in wild type (WT) plants. In addition, the grain volume of tasg1 was significantly higher than that of WT at the early filling stage. The content of various cytokinins (CKs) in the grain was significantly higher in tasg1 than in WT and was accompanied by an upregulated expression of some cell cycle-related genes. Examination of the metabolism of soluble sugars in tasg1 and WT revealed that the concentrations of glucose (Glu), fructose (Fru), and sucrose (Suc) were higher in the flag leaves and grains of tasg1 than in WT plants. The activities of sucrose-phosphate synthase (SPS), sucrose synthase (SuSy), and cell wall invertase (CW-invertase) were higher in tasg1, suggesting an altered metabolism and transport of soluble sugars. Furthermore, when tasg1 was treated with the CK inhibitor lovastatin, the activity of invertase was inhibited and was associated with premature senescence phenotype. However, the activity of invertase was partially recovered in tasg1 when treated with 6-benzylaminopurine (BAP). The trend of change in the concentrations of Glu, Fru, and Suc was similar to that of invertase. Our results suggest that CKs might regulate the stay-green phenotype of tasg1 by regulating the invertase activity involved in Suc remobilization. PMID:27580166

Nodulation is tightly regulated in legumes to ensure appropriate levels of nitrogen fixation without excessive depletion of carbon reserves. This balance is maintained by intimately linking nodulation and its regulation with plant hormones. It has previously been shown that ethylene and jasmonic acid (JA) are able to regulate nodulation and Nod factor signal transduction. Here, we characterize the nature of abscisic acid (ABA) regulation of nodulation. We show that application of ABA inhibits nodulation, bacterial infection, and nodulin gene expression in Medicago truncatula. ABA acts in a similar manner as JA and ethylene, regulating Nod factor signaling and affecting the nature of Nod factor-induced calcium spiking. However, this action is independent of the ethylene signal transduction pathway. We show that genetic inhibition of ABA signaling through the use of a dominant-negative allele of ABSCISIC ACID INSENSITIVE1 leads to a hypernodulation phenotype. In addition, we characterize a novel locus of M. truncatula, SENSITIVITY TO ABA, that dictates the sensitivity of the plant to ABA and, as such, impacts the regulation of nodulation. We show that ABA can suppress Nod factor signal transduction in the epidermis and can regulate cytokinin induction of the nodule primordium in the root cortex. Therefore, ABA is capable of coordinately regulating the diverse developmental pathways associated with nodule formation and can intimately dictate the nature of the plants' response to the symbiotic bacteria.

Full Text Available The germination of caryopses of M-T3 Triticale generation, which were freshly harvested in different growth and developmental phases has been studied. A significant influence of the abscisic acid (ABA accumulation on the increment of number of germinating caryopses has been found. Already in the first phase af the embryogenesis considerable stimulating effects of kinetin and gibberellin-A3 (GA3 on the germination of embryos which were isolated from freshly collected grains have been shown. When both stimulators were used together marked synergetic reaction occurred. It has been also determined that in the initial period of embryogensis premature germination occurs, to a higher extent, under the action of cytokinins than! Whether in the further phases of the caryopse development, when embryo develop mainly through the cell elongation, mostly gibberellins seem to be responsible for the activation of germination processes. The more mature were seeds the quicker germinated whole caryopses and embryos isolated from them at different ripeness, after 3-month storage. The highest stimulation of germination by phytohormones has been found for the most mature caryopses. The action of gibberellic acid has been particulary strong.

Based on the finding of a novel berberine (BBR)-utilizing bacterium, Rhodococcus sp. strain BD7100, we investigated the degradation of BBR and its analog berberrubine (BRU). Resting cells of BD7100 demethylenated BBR and BRU, yielding benzeneacetic acid analogs. Isolation of benzeneacetic acid analogs suggested that BD7100 degraded the isoquinoline ring of the protoberberine skeleton. This work represents the first report of cleavage of protoberberine skeleton by a microorganism.

Dibenzothiophene (DBT) monooxygenase (DszC)catalysis,the first and also the key step in the microbial DBT desulfurization,is the conversion of DBT to DBT sulfone (DBTO2).In this study,dszC of a DBT-desulfiaizing bacterium Rhodococcus sp.DS-3 was cloned by PCR.The sequence cloned was 99% homologous to Rhodococcus erythropolis IGTS8 that was reported in the Genebank.The gene dszC could be overexpressed effectively after being inserted into plasmid pET28a and transformed into E.coli BL21 strain.The expression amount of DszC was about 20% of total supernatant at low temperature.The soluble DszC in the supematant was purified by Ni2+ chelating His-Tag resin column and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to electronics purity.Only one band was detected by Western-blotting,which is for the antibody released in mouse against purified DszC in the expression product of BL21 (DE3,paC5) and Rhodococcus sp.DS-3.The activity of purified DszC was 0.36 U.DszC can utilize the organic compound such as DBT and methyl-DBT,hut not DBT derivates such as DBF,which has no sulfur or inorganic sulfur.

As oleaginous microorganisms represent an upcoming novel feedstock for the biotechnological production of lipids or lipid-derived biofuels, we searched for novel, lipid-producing strains in desert soil. This was encouraged by the hypothesis that neutral lipids represent an ideal storage compound, especially under arid conditions, as several animals are known to outlast long periods in absence of drinking water by metabolizing their body fat. Ten lipid-accumulating bacterial strains, affiliated to the genera Bacillus, Cupriavidus, Nocardia, Rhodococcus and Streptomyces, were isolated from arid desert soil due to their ability to synthesize poly(β-hydroxybutyrate), triacylglycerols or wax esters. Particularly two Streptomyces sp. strains and one Rhodococcus sp. strain accumulate significant amounts of TAG under storage conditions under optimized cultivation conditions. Rhodococcus sp. A27 and Streptomyces sp. G49 synthesized approx. 30% (w/w) fatty acids from fructose or cellobiose, respectively, while Streptomyces isolate G25 reached a cellular fatty acid content of nearly 50% (w/w) when cultivated with cellobiose. The stored triacylglycerols were composed of 30-40% branched fatty acids, such as anteiso-pentadecanoic or iso-hexadecanoic acid. To date, this represents by far the highest lipid content described for streptomycetes. A biotechnological production of such lipids using (hemi)cellulose-derived raw material could be used to obtain sustainable biodiesel with a high proportion of branched-chain fatty acids to improve its cold-flow properties and oxidative stability.

Full Text Available Introduction Drought is one of the major environmental conditions that adversely affects plant growth and crop yield. In the face of a global scarcity of water resources, water stress has already become a primary factor in limiting crop production worldwide. Drought is the major restriction in maize production. The plant growth reduction under drought stress conditions could be an outcome of altered hormonal balance and hence the exogenous application of growth regulators under stress conditions could be the possible means for reversing the effects of abiotic stress. Phytohormones such as auxine and cytokinine are known to be involved in the regulation of plant response to the adverse effects of stress conditions. Previous studies have shown that endogenous hormones are essential regulators for translocation and partitioning of photoassimilates for grain filling in cereal crops, and therefore could be involved in the regulation of grain weight and yield. Materials and Methods The experiment was carried out in three separately environments included non-drought stress environment (irrigation after soil moisture reached to 75% field capacity, drought stress in vegetative stage (irrigation after soil moisture reached to 50% field capacity in V4 to tasseling stage, but irrigation after soil moisture reached to 75% field capacity in pollination to physiological maturity stage and drought stress in reproductive stage (irrigation after soil moisture reached to 75% field capacity in V4 to tasseling stage and irrigation after soil moisture reached to 50% field capacity in pollination to physiological maturity stage. Cytokinin hormone in three levels (control, spraying in V5 –V6 and V8-V10 stages and auxin hormone in three levels (control, spraying in silk emergence stage and 15 days after that were laid out as a factorial design based on randomized complete block with three replications in each environment at Seed and Plant Improvement Institute (SPII

Full Text Available Effects Oif adenosdne-3',5'-cyclic monophosphate (cAMP, N6,O2-dibuityryl adenosine-3',5'-cyclic monophosphate (DBcAMP, caffeine and theophylline on the bud-inducing activity of cytokinin in the protonema of two moss species, Ceratodon purpureus and Funaria hygrometrica were examined. The sub-stances have been found ineffective as gametophore bud inducers. Some synergism between cytokinin and cAMP or DBcAMP was observed with relation to the buds' growth, but this effect is nonspecific since it can be obtained with 5'-AMP or 5'-GMiP as well, The results seem to exclude the possibility of an involvement of cAMP as a second messenger in the mechanism of cytokinin action on morphogenetic processes in moss protonemata.

Post-embedding immunocytochemical techniques using peroxidase-antiperoxidase as markers for light microscopy or immunoglobulin G-gold for electron microscopy respectively were used for the localization of cytokinins [9-beta-D-ribofuranosil-N6-(delta2-isopentenil) adenina ([9R]iP), 9-beta-D-ribofuranosyl-zeatin ([9R]Z) and 9-beta-D-ribofuranosyl-dihydrozeatin ([9R](diH)Z)] in kiwifruit (Actinidia deliciosa) meristematic cells of the second nodal segment. Immunolocation at the cellular level was carried out in cells from explants grown during 16 and 72 h in liquid medium. Subcellular immunolocalization was performed in cells from explants grown for 35 d on agar solidified-medium and for 30 min, 4 and 16 h in liquid medium with cellulose plugs as explant support. Taken as a whole, the results obtained for Actinidia deliciosa show that the studied cytokinins change their location during the culture period, although they can always be found to a greater or lesser extent in the nucleus and the cytoplasm. For instance, [9R]Z appears in the cytoplasm and in the nucleus during the first hours of culture and later is the only one that appears located mainly in nucleus. On the other hand, [9R](diH)Z changes from being predominantly located in the nucleus to practically appearing only in the cytoplasm at the end of the culture period. [9R]iP is principally found up to 4 h of culture in the cytoplasm, and at 16 h is evenly distributed in all the subcellular compartments except in the chloroplast. The existence of a large amount of cytokinins in the nucleus during the first hours of culture compared with the immunolabelling density at 35 d is probably due to the activation of cell cycle mechanisms leading to organogenic development at the beginning of culture.

Cytokinin signaling is vital for plant growth and development which function via the two-component system (TCS). As one of the key component of TCS, transmembrane histidine kinases (HK) are encoded by a small gene family in plants. In this study, we focused on expression and functional analysis of cytokinin receptor-like HK genes (ZmHK) in maize. Firstly, bioinformatics analysis revealed that seven cloned ZmHK genes have different expression patterns during maize development. Secondly, ectopic expression by CaMV35S promoter in Arabidopsis further revealed that functional differentiation exists among these seven members. Among them, the ZmHK1a2-OX transgenic line has the lowest germination rate in the dark, ZmHK1-OX and ZmHK2a2-OX can delay leaf senescence, and seed size of ZmHK1-OX, ZmHK1a2-OX, ZmHK2-OX, ZmHK3b-OX and ZmHK2a2-OX was obviously reduced as compared to wild type. Additionally, ZmHK genes play opposite roles in shoot and root development; all ZmHK-OX transgenic lines display obvious shorter root length and reduced number of lateral roots, but enhanced shoot development compared with the wild type. Most notably, Arabidopsis response regulator ARR5 gene was up-regulated in ZmHK1-OX, ZmHK1a2-OX, ZmHK2-OX, ZmHK3b-OX and ZmHK2a2-OX as compared to wild type. Although the causal link between ZmHK genes and cytokinin signaling pathway is still an area to be further elucidated, these findings reflected that the diversification of ZmHK genes expression patterns and functions occurred in the course of maize evolution, indicating that some ZmHK genes might play different roles during maize development.

The plant phytohormone cytokinin plays an important role in many facets of plant growth and development by regulating cell division and differentiation. Recent studies have shed significant light into the mechanisms of cytokinin metabolism and signaling. However, little is known about how the hormone is transported in planta, although it has been proposed that the hormone is presumably transported in nucleoside-conjugated forms. Here, we report the identification and characterization of cytokinin transport ers in Arabidopsis. We previously reported that a gain-of-function mutation in the PGA22/AtIPT8 gene caused overproduction of cytokinins in planta. In an effort to screen for suppressor of pga22/atipt8 (soi) mutants, we identified a mutant soi33-1. Molecular and genetic analyses indicated that SOI33 encodes a putative equilibrative nucleoside transporter (ENT), previously designated as AtENT8. Members of this small gene family are presumed to be involved in the transport of nucleosides in eukaryotic cells. Under conditions of nitrogen starvation, loss-of-function mutations in SOI33/AtENT8 or in a related gene AtENT3 cause a reduced sensitivity to the nucleoside-type cytokinins isopentenyladenine riboside (iPR) and trans zeatin riboside (tZR), but display a normal response to the free base-type cytokinins isopentenyladenine (iP) and trans-zeatin (tZ). Conversely, overexpression of SOI33/AtENT8 renders transgenic plants hyper sensitive to iPR but not to iP. An in planta measurement experiment indicated that uptake efficiency of 3H labeled iPR was reduced more than 40% in soi33 and atent3 mutants. However, a mutation inAtENT1 had no substantial effect on the cytokinin response and iPR uptake efficiency. Our results suggest that SOI33/ AtENT8 and AtENT3 are involved in the transport of nucleoside-type cytokinins in Arabidopsis.

The soil-dwelling, saprophytic actinomycete Rhodococcus equi is a multihost, facultative intracellular pathogen of macrophages. When inhaled by susceptible foals, it causes severe bronchopneumonia. It is also a pathogen of pigs, which may develop submaxillary lymphadenitis upon exposure. R. equi isolates obtained from foals and pigs possess conjugative plasmids housing a pathogenicity island (PAI) containing a novel family of genes of unknown function called the virulence-associated protein or vap family. The PAI regions of the equine and swine plasmids differ in vap gene composition, with equine isolates possessing six vap genes, including the major virulence determinant vapA, while the PAIs of swine isolates house vapB and five other unique vap genes. Possession of the pVAPA-type virulence plasmid by equine isolates bestows the capacity for intramacrophage replication essential for disease development in vivo. Swine isolates of R. equi are largely unstudied. Here, we show that R. equi isolates from pigs, carrying pVAPB-type plasmids, are able to replicate in a plasmid-dependent manner in macrophages obtained from a variety of species (murine, swine, and equine) and anatomical locations. Similarly, equine isolates carrying pVAPA-type plasmids are capable of replication in swine macrophages. Plasmid swapping between equine and swine strains through conjugation did not alter the intracellular replication capacity of the parental strain, indicating that coevolution of the plasmid and chromosome is not crucial for this attribute. These results demonstrate that while distinct plasmid types exist among R. equi isolates obtained from equine and swine sources, this tropism is not determined by host species-specific intramacrophage replication capabilities. IMPORTANCE This work greatly advances our understanding of the opportunistic pathogen Rhodococcus equi, a disease agent of animals and immunocompromised people. Clinical isolates from diseased foals carry a

Cytokinin (CK) is a vital plant hormone that controls many aspects of growth and development in plants. Nitrogen (N) is the indispensable macronutrient needed in plants and also one of the most important limiting factors for plant growth. This study was designed to investigate the simultaneous effects of CK and N on the visual turf quality and antioxidant metabolism of drought-stressed creeping bentgrass (Agrostis stolonifera L.). 'PennA-4' creeping bentgrass treated with trans-zeatin riboside at three rates of CK concentrations of 0, 10 and 100 μM (designated by CK0, 10, and 100) and two nitrogen rates with 2.5 and 7.5 kg N·ha-1 every 15 days (designated by low and high N) in a complete factorial arrangement was grown under two soil moisture regimes: well-watered and drought stress. Exogenous CK improved turf quality and delayed leaf wilting under drought stress, especially under high N. The grasses treated with CK10 and CK100 had lower O2- production and H2O2 concentration than those without CK treatment. The CK100 treatment enhanced the activities of superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), and guaiacol peroxidase (POD) by 25%, 22%, 17% and 24%, respectively, relative to CK0. Moreover, the activity changes of the antioxidant enzyme isoforms were more significant under high N condition relative to low N condition. Our results demonstrated the beneficial impacts of CK and N on physiological reactions, especially antioxidant metabolism, and foliar application of CK at 10 or 100 μM plus 7.5 kg ha-1 N biweekly may improve drought stress resistance of creeping bentgrass.

Recently, a renewed interest in cytokinins (CKs) has allowed the characterization of these phytohormones as key regulatory molecules in plant biotic interactions. They have been proved to be instrumental in microbe- and insect-mediated plant phenotypes that can be either beneficial or detrimental for the host-plant. In parallel, insect endosymbiotic bacteria have emerged as key players in plant-insect interactions mediating directly or indirectly fundamental aspects of insect nutrition, such as insect feeding efficiency or the ability to manipulate plant physiology to overcome food nutritional imbalances. However, mechanisms that regulate CK production and the role played by insects and their endosymbionts remain largely unknown. Against this backdrop, studies on plant-associated bacteria have revealed fascinating and complex molecular mechanisms that lead to the production of bacterial CKs and the modulation of plant-borne CKs which ultimately result in profound metabolic and morphological plant modifications. This review highlights major strategies used by plant-associated bacteria that impact the CK homeostasis of their host-plant, to raise parallels with strategies used by phytophagous insects and to discuss the possible role played by endosymbiotic bacteria in these CK-mediated plant phenotypes. We hypothesize that insects employ a CK-mix production strategy that manipulates the phytohormonal balance of their host-plant and overtakes plant gene expression causing a metabolic and morphological habitat modification. In addition, insect endosymbiotic bacteria may prove to be instrumental in these manipulations through the production of bacterial CKs, including specific forms that challenge the CK-degrading capacity of the plant (thus ensuring persistent effects) and the CK-mediated plant defenses.

The present study was conducted to determine the prevalence of Rhodococcus equi infection in equines of Jammu and Kashmir, India, and evaluate the zoonotic threat posed by this organism to equine owners and tourists. One hundred and forty-one samples (98 samples from adult animals ≥5 years old and 43 samples from foals less than 6 months old) were collected in duplicate from nasopharyngeal tract of equines for isolation and direct PCR. A total of 12 isolates of R. equi were recovered, of which 9 were from foals and 3 from adult animals. Therefore, the present study recorded prevalence rates of 20.93% and 3.06% among foals and adult equines respectively. The prevalence rates were found to be 25.58% and 4.08% by 16S rRNA species-specific PCR among foals and adult animals respectively. Thus, the PCR-based assay was found to be more sensitive and helped in quick detection of R. equi than the culture based method which is time consuming and laborious. However, the culture-based method is still preferred due to some limitations of PCR. The antibiogram of the isolates revealed that erythromycin and rifampicin were the most effective antimicrobials with 100% sensitivity, followed by amoxicillin (66.67%), lincomycin (58.3%) and kanamycin (58.3%). The results also revealed that resistance was highest for penicillin G (50%), followed by kanamycin (25%) and streptomycin (25%).

Advanced biofuels from lignocellulosic biomass have been considered as a potential solution for the issues of energy sustainability and environmental protection. Triacylglycerols (TAGs) are potential precursors for the production of lipid-based liquid biofuels. Rhodococcus opacus PD630 can accumulate large amounts of TAGs when grown under physiological conditions of high carbon and low nitrogen. However, R. opacus PD630 does not utilize the sugar L-arabinose present in lignocellulosic hydrolysates. Here, we report the engineering of R. opacus to produce TAGs on L-arabinose. We constructed a plasmid (pASC8057) harboring araB, araD and araA genes derived from a Streptomyces bacterium, and introduced the genes into R. opacus PD630. One of the engineered strains, MITAE-348, was capable of growing on high concentrations (up to 100 g/L) of L-arabinose. MITAE-348 was grown in a defined medium containing 16 g/L L-arabinose or a mixture of 8 g/L L-arabinose and 8 g/L D-glucose. In a stationary phase occurring 3 days post-inoculation, the strain was able to completely utilize the sugar, and yielded 2.0 g/L for L-arabinose and 2.2 g/L for L-arabinose/D-glucose of TAGs, corresponding to 39.7% or 42.0%, respectively, of the cell dry weight.

Rhodococcus equi causes a rare infection in immunocompromised hosts. We describe 24 cases of infection in patients with AIDS-related complex (ARC)/acquired immunodeficiency syndrome (AIDS). Pneumonia was always the first manifestation of R. equi infection, but extrapulmonary involvement was also observed. The main sources of bacteria were sputum, bronchial washings and blood. The strains isolated were mainly susceptible to erythromycin, vancomycin, teicoplanin, rifampicin, imipenem and aminoglycosides. Initial treatment should involve an intravenously administered antibiotic combination therapy including imipenem or vancomycin or teicoplanin, followed by orally administered maintenance combination therapy. Drug combinations should be investigated for serum bactericidal activity in vitro. Surgery does not increase survival time and should only be performed in cases that do not respond to antibiotic treatment. Presumptive risks of infection (contact with horses or farm dust, or cohabiting with people affected by R. equi infection) were present in more than 50% of patients. This finding, and the frequency of bacteria in the sputum, are not sufficient proof of transmission between humans, but do suggest the need for respiratory isolation of patients affected by R. equi pneumonia.

Metabolic pathways of aerobic bacteria able to assimilate sulfur can provide biocatalysts for biodesulfurization of petroleum and of other sulfur-containing pollutants. Of major interest is the so-called "4S pathway," in that C-S bonds are specifically cleaved leaving the carbon skeleton of substrates intact. This pathway is carried out by four enzymes, named Dsz A, B, C, and D. In view of a possible application of recombinant Dsz enzymes in biodesulfurization treatments, we have investigated the structural features of enzymes cloned from a Rhodococcus strain isolated from polluted environmental samples and their resistance to temperature (20-95 °C) and to organic solvents (5, 10, and 20 % v/v methanol, acetonitrile, hexane, and toluene). Changes in protein structures were assessed by circular dichroism and intrinsic fluorescence spectroscopy. We found that all Dsz proteins are unfolded by temperatures in the range 45-60 °C and by all solvents tested, with the most dramatic effect being produced by toluene. These results suggest that stabilization of the biocatalysts by protein engineering will be necessary for developing biodesulfurization technologies based on Dsz enzymes.

Rhodococcus erythropolis LSSE8-1 is a newly isolated biodesulfurizaion strain from the soil of Chishui gas field, Guizhou Province, China. The analysis of its metabolism product shows that the strain is a kind of biocatalyst able to oxidize dibenzothiophene (DBT) to 2-hydroxydi- phenyl (HBP), and therefore the sulfur in DBT is selectively removed. By using DBTO2 (dibenzothiophene 5,5-dioxide) as substrate, both DBT and HBP are found in the culture, which shows that the reaction from DBT to DBTO2 is reversible in the cell. While using 0.5 mmol/L DBT as control, 0.01-0.4 mmol/L DBTO2 shows poisonous effect to the cell, which will explain why there is no DBTO2 accumulation in the process of biodesulfurization. After treatment by lysozme, the plasmid DNA of the strain is isolated by alkaline method to be used as the template of PCR reaction. Three dsz gene fragments of 1.3, 1.0 and 1.2 kb respectively were amplified. Each fragment is ligate with PGEM-T vector, and cloned into E. coli. DH5α. The clone DNA is sequenced and the result shows that dsz related genes are highly conservative. The identities of dszA and dszB with respect to IGTS8 are 100%, and the identity of dszC with that of IGTS8 is 99%.

Full Text Available Abstract Recently, there has been a lot of interest in the utilization of rhodococci in the bioremediation of petroleum contaminated environments. This study investigates the response of Rhodococcus erythropolis IBBPo1 cells to 1% organic solvents (alkanes, aromatics. A combination of microbiology, biochemical, and molecular approaches were used to examine cell adaptation mechanisms likely to be pursued by this strain after 1% organic solvent exposure. R. erythropolis IBBPo1 was found to utilize 1% alkanes (cyclohexane, n-hexane, n-decane and aromatics (toluene, styrene, ethylbenzene as the sole carbon source. Modifications in cell viability, cell morphology, membrane permeability, lipid profile, carotenoid pigments profile and 16S rRNA gene were revealed in R. erythropolis IBBPo1 cells grown 1 and 24 h on minimal medium in the presence of 1% alkanes (cyclohexane, n-hexane, n-decane and aromatics (toluene, styrene, ethylbenzene. Due to its environmental origin and its metabolic potential, R. erythropolis IBBPo1 is an excellent candidate for the bioremediation of soils contaminated with crude oils and other toxic compounds. Moreover, the carotenoid pigments produced by this nonpathogenic Gram-positive bacterium have a variety of other potential applications.

Full Text Available Abstract Background Rhodococcus equi (R.equi is an acid fast, GRAM + coccobacillus, which is widespread in the soil and causes pulmonary and extrapulmonary infections in immunocompromised people. In the context of HIV infection, R.equi infection (rhodococcosis is regarded as an opportunistic disease, and its outcome is influenced by highly active antiretroviral therapy (HAART. Case presentation We report two cases of HIV-related rhodococcosis that disseminated despite suppressive HAART and anti-rhodococcal treatment; in both cases there was no immunological recovery, with CD4+ cells count below 200/μL. In the first case, pulmonary rhodococcosis presented 6 months after initiation of HAART, and was followed by an extracerebral intracranial and a cerebral rhodococcal abscess 1 and 8 months, respectively, after onset of pulmonary infection. The second case was characterized by a protracted course with spread of infection to various organs, including subcutaneous tissue, skin, colon and other intra-abdominal tissues, and central nervous system; the spread started 4 years after clinical resolution of a first pulmonary manifestation and progressed over a period of 2 years. Conclusions Our report highlights the importance of an effective immune recovery, despite fully suppressive HAART, along with anti-rhodococcal therapy, in order to clear rhodococcal infection.

An agar-degrading bacterium, Rhodococcus sp. Q5, was isolated from printing and dyeing wastewater using a mineral salts agar plate containing agar as the sole carbon source. The bacterium grew from pH 4.0 to 9.0, from 15 to 35°C, and in NaCl concentrations of 0-5 %; optimal values were pH 6.0, 30°C, and 1 % NaCl. Maximal agarase production was observed at pH 6.0 and 30°C. The bacterium did not require NaCl for growth or agarase production. The agarase secreted by Q5 was inducible by agar and was repressed by all simple sugars tested except lactose. Strain Q5 could hydrolyze starch but not cellulose or carboxymethyl cellulose. Agarase activity could also be detected in the medium when lactose or starch was the sole source of carbon and energy. Strain Q5 could grow in nitrogen-free mineral media; an organic nitrogen source was more effective than inorganic carbon sources for growth and agarase production. Addition of more organic nitrogen (peptone) to the medium corresponded with reduced agarase activity.

In most habitats, the vast majority of microbial populations form biofilms on solid surfaces, whether natural or artificial. These biofilms provide either increased physical support and/or a source of nutrients. Further modifications and development of biofilms are regulated by signal molecules secreted by the cells. Because synthetic polymers are not soluble in aqueous solutions, biofilm-producing bacteria may biodegrade such materials more efficiently than planktonic strains. Bacterial biofilms comprise bacterial cells embedded in self-secreted extracellular polymeric substances (EPS). Revealing the roles of each component of the EPS will enable further insight into biofilm development and the EPS structure-function relationship. A strain of Rhodococcus ruber (C208) displayed high hydrophobicity and formed a dense biofilm on the surface of polyethylene films while utilizing the polyolefin as carbon and energy sources. This study investigated the effects of several proteases on C208 biofilm formation and stability. The proteolysis of C208 biofilm gave conflicting results. Trypsin significantly reduced biofilm formation, and the resultant biofilm appeared monolayered. In contrast, proteinase K enhanced biofilm formation, which was robust and multilayered. Presumably, proteinase K degraded self-secreted proteases or quorum-sensing peptides, which may be involved in biofilm detachment processes, leading to a multilayered, nondispersed biofilm.

A Rhodococcus species, which has proven to be the best of 99 oil-emulsifying bacteria isolated from seawater, was characterized. This bacterium produced very stable oil-in-water emulsions from different crude oils with various content of aliphatic and aromatic compounds, by utilizing C{sub 1}1 and C{sub 3}3 n-alkanes as carbon and energy sources. Bacteria that produce stable emulsions are often able to adhere strongly to hydrocarbons or hydrophobic surfaces. It was at these surfaces that extensive emulsification of the residual oil and accumulation of acidic oxidation products occurred. The acidic products were consumed in a second step. This step was characterized by linear growth and an increasing number of cells growing in the water phase. The most extensive emulsification occurred at the end of the exponential phase. There was no evidence of surfactants at the end of the exponential phase, however, a polymeric compound with emulsifying activity, tightly bound to the oil droplets, was isolated, suggesting that the emulsification resulted from the release of the hydrophobic cell surface discarded during growth limitations. 38 refs., 7 figs.

Rhodococcus jostii RHA1 (RHA1) degrades polychlorinated biphenyl (PCB) via co-metabolism with biphenyl. To identify the novel open reading frames (ORFs) that contribute to PCB/biphenyl metabolism in RHA1, we compared chromatin immunoprecipitation chip and transcriptomic data. Six novel ORFs involved in PCB/biphenyl metabolism were identified. Gene deletion mutants of these 6 ORFs were made and were tested for their ability to grow on biphenyl. Interestingly, only the ro10225 deletion mutant showed deficient growth on biphenyl. Analysis of Ro10225 protein function showed that growth of the ro10225 deletion mutant on biphenyl was recovered when exogenous recombinant Ro10225 protein was added to the culture medium. Although Ro10225 protein has no putative secretion signal sequence, partially degraded Ro10225 protein was detected in conditioned medium from wild-type RHA1 grown on biphenyl. This Ro10225 fragment appeared to form a complex with another PCB/biphenyl oxidation enzyme. These results indicated that Ro10225 protein is essential for the formation of the PCB/biphenyl dioxygenase complex in RHA1.

The nhhBAG gene of Rhodococcus rhodochrous M33 that encodes nitrile hydratase (NHase), converting acrylonitrile into acrylamide, was cloned and expressed in Corynebacterium glutamicum under the control of an ilvC promoter. The specific enzyme activity in recombinant C. glutamicum cells was about 13.6 μmol/min/mg dry cell weight (DCW). To overexpress the NHase, five types of plasmid variants were constructed by introducing mutations into 80 nucleotides near the translational initiation region (TIR) of nhhB. Of them, pNBM4 with seven mutations showed the highest NHase activity, exhibiting higher expression levels of NhhB and NhhA than wild-type pNBW33, mainly owing to decreased secondary-structure stability and an introduction of a conserved Shine-Dalgarno sequence in the translational initiation region. In a fed-batch culture of recombinant Corynebacterium cells harboring pNBM4, the cell density reached 53.4 g DCW/L within 18 h, and the specific and total enzyme activities were estimated to be 37.3 μmol/min/mg DCW and 1,992 μmol/min/mL, respectively. The use of recombinant Corynebacterium cells for the production of acrylamide from acrylonitrile resulted in a conversion yield of 93 % and a final acrylamide concentration of 42.5 % within 6 h when the total amount of fed acrylonitrile was 456 g.

Rhodococcus sp. BTO62 was isolated from activated sludge from a wastewater treatment plant as an o-xylene-degrading microorganism. BOT62 degraded not only o-xylene, but also benzene, toluene, ethylbenzene, m- and p-xylenes and styrene (BTEXS). A laboratory scale biofilter packed with Biosol as packing material, which is made from foamed waste glass mixed with corrugated cardboard, was inoculated with strain BTO62 and operated to remove relatively high loading of o-xylene at different space velocities under non-sterile and sterile conditions. The o-xylene elimination capacity to maintain more than 90% removal efficiency was 41g/m3/h under sterile condition, but it enhanced to 160g/m3/h under non-sterile condition. This indicates possibilities of the role of other contaminants for degradation of o-xylene and the degradation of intermediate products of o-xylene by contaminants. Quick recovery of o-xylene degradation was observed after shutdown of o-xylene gas supply and mineral medium circulation for 10-30 days.

A new acylamidase was isolated from Rhodococcus erythropolis TA37 and characterized. N-Substituted acrylamides (isopropyl acrylamide, N,N-dimethyl-aminopropyl acrylamide, and methylene-bis-acrylamide), acid para-nitroanilides (4'-nitroacetanilide, Gly-pNA, Ala-pNA, Leu-pNA), and N-acetyl derivatives of glycine, alanine, and leucine are good substrates for this enzyme. Aliphatic amides (acetamide, acrylamide, isobutyramide, n-butyramide, and valeramide) are also used as substrates but with less efficiency. The enzyme subunit mass by SDS-PAGE is 55 kDa. Maximal activity is exhibited at pH 7-8 and 55°C. The enzyme is stable for 15 h at 22°C and for 0.5 h at 45°C. The Michaelis constant (K(m)) is 0.25 mM with Gly-pNA and 0.55 mM with Ala-pNA. The acylamidase activity is suppressed by inhibitors of serine proteases (phenylmethylsulfonyl fluoride and diisopropyl fluorophosphate) but is not suppressed by inhibitors of aliphatic amidases (acetaldehyde and nitrophenyl disulfides). The N-terminal amino acid sequence of the acylamidase is highly homologous to those of two putative amidases detected from sequenced R. erythropolis genomes. It is suggested that the acylamidase together with the detected homologs forms a new class within the amidase signature family.

Rhodococcus equi is an intracellular pathogen which causes pneumonia in young horses and in immunocompromised humans. R. equi arrests phagosome maturation in macrophages at a prephagolysosome stage and grows inside a privileged compartment. Here, we show that, in murine macrophages activated with gamma interferon and lipopolysaccharide, R. equi does not multiply but stays viable for at least 24 h. Whereas infection control of other intracellular pathogens by activated macrophages is executed by enhanced phagosome acidification or phagolysosome formation, by autophagy or by the interferon-inducible GTPase Irgm1, none of these mechanisms seems to control R. equi infection. Growth control by macrophage activation is fully mimicked by treatment of resting macrophages with nitric oxide donors, and inhibition of bacterial multiplication by either activation or nitric oxide donors is annihilated by cotreatment of infected macrophages with ferrous sulfate. Transcriptional analysis of the R. equi iron-regulated gene iupT demonstrates that intracellular R. equi encounters iron stress in activated, but not in resting, macrophages and that this stress is relieved by extracellular addition of ferrous sulfate. Our results suggest that nitric oxide is central to the restriction of bacterial access to iron in activated macrophages.

Rhodococcus equi is a gram positive coccoid rod that causes pulmonary infections in immunosuppressed patients. We retrospectively analyzed epidemiological, clinical, microbiological, radiological, and immunological features as well as the outcomes of 13 AIDS patients with R. equi infection. Between January 1994 and December 2012, 13 patients attending the AIDS department of the Infectious Diseases reference hospital in Buenos Aires were diagnosed with R. equi infection. All were men, the median age was 27 years. At the time of diagnosis, the median of CD4+ T cell counts was 11 cells/μl Twelve patients presented pulmonary disease with isolation of the microorganism from sputum or bronchoalveolar lavage; in the other patient the diagnosis was postmortem with positive culture of cerebrospinal fluid. The most frequent clinical manifestations were fever, haemoptysis, and weight loss. The predominant radiological finding was lobe consolidation with cavitation. Nine patients died after a median survival of 5.5 months. In all of them, cultures persisted positive until the last admission. The other 4 patients did continue clinical follow-ups. The insidious course of R. equi disease and the difficulties in the isolation of the microorganism contribute to the delay in the diagnosis and to the high mortality rate of this opportunistic infection.

Full Text Available Rhodococcus equi is an important pathogen of foals that causes severe pneumonia. To date, there is no licensed vaccine effective against R. equi pneumonia of foals. The objectives of our study were to develop an electron beam (eBeam inactivated vaccine against R. equi and evaluate its immunogenicity. A dose of eBeam irradiation that inactivated replication of R. equi while maintaining outer cell wall integrity was identified. Enteral administration of eBeam inactivated R. equi increased interferon-γ production by peripheral blood mononuclear cells in response to stimulation with virulent R. equi and generated naso-pharyngeal R. equi-specific IgA in newborn foals. Our results indicate that eBeam irradiated R. equi administered enterally produce cell-mediated and upper respiratory mucosal immune responses, in the face of passively transferred maternal antibodies, similar to those produced in response to enteral administration of live organisms (a strategy which previously has been documented to protect foals against intrabronchial infection with virulent R. equi. No evidence of adverse effects was noted among vaccinated foals.

Full Text Available Abstract: Rhodococcus equi is a facultative intracellular pathogen, which cause severe pyogranulomatous pneumonia in foals and tuberculosis-like lesions in humans. Its ability to form biofilm was described in strains isolated from chronic diseases associated to treatment failures in humans. This study aimed to verify the biofilm formation by 113 R. equi isolated from equine samples (clinical and fecal using two different methods (biofilm-culturing with and without additional glucose and epifluorescence microscopy. We also aimed to determine the efficacy of azithromycin, clarithromycin and erythromycin on R. equi in established biofilm. We found 80.5% (26/41 and 63% (58/72 biofilm-positive isolates, in fecal and clinical samples, respectively. The additional glucose increased the biofilm formation by R. equi fecal samples, but not by clinical samples. The antimicrobials tested herein were not able to eradicate R. equi in biofilm even at higher concentrations. This is the ﬁrst study showing the biofilm formation by R. equi isolated from equine samples. Our findings indicate that R. equi biofilm-producers may be more resistant to the antimicrobials evaluated. Further studies are warranted to test this hypothesis.

Full Text Available Abstract Introduction Rhodococcus equi is an animal pathogen that was initially isolated from horses and is being increasingly reported as a cause of infection in humans with impaired cellular immunity. However, this pathogen is underestimated as a challenging antagonist and is frequently considered to be a mere contaminant despite the potential for life-threatening infections. Most case reports have occurred in immunocompromised patients who have received organ transplants (for example kidney, heart, bone marrow or those with human immunodeficiency virus infection. Infections often manifest as pulmonary involvement or soft tissue abscesses. Bacteremia related to R. equi infections of tunneled central venous catheters has rarely been described. Case presentation We report the case of a 63-year-old non-transplant recipient, non-HIV infected Caucasian woman with endometrial carcinoma who developed recurrent bloodstream infections and septic shock due to R. equi and ultimately required the removal of her port catheter, a subcutaneous implantable central venous catheter. We also review the medical literature related to human infections with R. equi. Conclusion R. equi should be considered a serious pathogen, not a contaminant, particularly in an immunocompromised patient who presents with a central venous catheter-related bloodstream infection. Counseling patients with central venous catheters who participate in activities involving exposure to domesticated animals is recommended.

Rhodococcus equi is an opportunistic human pathogen associated with immunosuppressed people. While the interaction of R. equi with macrophages has been comprehensively studied, little is known about its interactions with non-phagocytic cells. Here, we characterized the entry process of this bacterium into human lung epithelial cells. The invasion is inhibited by nocodazole and wortmannin, suggesting that the phosphatidylinositol 3-kinase pathway and microtubule cytoskeleton are important for invasion. Pre-incubation of R. equi with a rabbit anti-R. equi polyclonal antiserum resulted in a dramatic reduction in invasion. Also, the invasion process as studied by immunofluorescence and scanning electron microscopy indicates that R. equi make initial contact with the microvilli of the A549 cells, and at the structural level, the entry process was observed to occur via a zipper-like mechanism. Infected lung epithelial cells upregulate the expression of cytokines IL-8 and IL-6 upon infection. The production of these pro-inflammatory cytokines was significantly enhanced in culture supernatants from cells infected with non-mucoid plasmid-less strains when compared with cells infected with mucoid strains. These results demonstrate that human airway epithelial cells produce pro-inflammatory mediators against R. equi isolates.

Full Text Available The gram-positive bacterium Rhodococcus equi was isolated from fertile soil, and mineral salt media (MM and trace elements were used to provide the necessary elements for its growth and PHB production in addition to using crude palm kernel oil (CPKO 1% as the carbon source. Gas chromatography (GC demonstrated that the composition of the recovered biopolymer was homopolymer polyhydroxybutyrate (PHB. The strain of the present study has a dry biomass of 1.43 (g/l with 38% PHB, as determined by GC. The recovered PHB was characterized by NMR to study the chemical structure. In addition, DSC and TGA were used to study the thermal properties of the recovered polymer, where the melting temperature (Tm was 173 °C, the glass transition temperature (Tg was 2.79 °C, and the decomposition temperature (Td was 276 °C. Gel permeation chromatography (GPC was used to study the molecular mass of the recovered PHB in addition to comparing the results with other studies using different bacteria and substrates, where the molecular weight was 642 kDa, to enable its usage in many applications. The present study demonstrated the use of an inexpensive substrate for PHB production, i.e., using gram-positive bacteria to produce PHB polymer with characterization.

Rhodococcus equi is an intracellular pathogen which causes pneumonia in young horses and in immunocompromised humans. R. equi arrests phagosome maturation in macrophages at a prephagolysosome stage and grows inside a privileged compartment. Here, we show that, in murine macrophages activated with gamma interferon and lipopolysaccharide, R. equi does not multiply but stays viable for at least 24 h. Whereas infection control of other intracellular pathogens by activated macrophages is executed by enhanced phagosome acidification or phagolysosome formation, by autophagy or by the interferon-inducible GTPase Irgm1, none of these mechanisms seems to control R. equi infection. Growth control by macrophage activation is fully mimicked by treatment of resting macrophages with nitric oxide donors, and inhibition of bacterial multiplication by either activation or nitric oxide donors is annihilated by cotreatment of infected macrophages with ferrous sulfate. Transcriptional analysis of the R. equi iron-regulated gene iupT demonstrates that intracellular R. equi encounters iron stress in activated, but not in resting, macrophages and that this stress is relieved by extracellular addition of ferrous sulfate. Our results suggest that nitric oxide is central to the restriction of bacterial access to iron in activated macrophages. PMID:21383050

Rhodococcus equi is an important pathogen of foals that causes severe pneumonia. To date, there is no licensed vaccine effective against R. equi pneumonia of foals. The objectives of our study were to develop an electron beam (eBeam) inactivated vaccine against R. equi and evaluate its immunogenicity. A dose of eBeam irradiation that inactivated replication of R. equi while maintaining outer cell wall integrity was identified. Enteral administration of eBeam inactivated R. equi increased interferon-γ production by peripheral blood mononuclear cells in response to stimulation with virulent R. equi and generated naso-pharyngeal R. equi-specific IgA in newborn foals. Our results indicate that eBeam irradiated R. equi administered enterally produce cell-mediated and upper respiratory mucosal immune responses, in the face of passively transferred maternal antibodies, similar to those produced in response to enteral administration of live organisms (a strategy which previously has been documented to protect foals against intrabronchial infection with virulent R. equi). No evidence of adverse effects was noted among vaccinated foals.

Rhodococcus equi is a multi-host pathogen that infects a range of animals as well as immune-compromised humans. Equine and porcine isolates harbour a virulence plasmid encoding a homologous family of virulence-associated proteins associated with the capacity of R. equi to divert the normal processes of endosomal maturation, enabling bacterial survival and proliferation in alveolar macrophages. To provide a basis for probing the function of the Vap proteins in virulence, the crystal structure of VapD was determined. VapD is a monomer as determined by multi-angle laser light scattering. The structure reveals an elliptical, compact eight-stranded β-barrel with a novel strand topology and pseudo-twofold symmetry, suggesting evolution from an ancestral dimer. Surface-associated octyl-β-D-glucoside molecules may provide clues to function. Circular-dichroism spectroscopic analysis suggests that the β-barrel structure is preceded by a natively disordered region at the N-terminus. Sequence comparisons indicate that the core folds of the other plasmid-encoded virulence-associated proteins from R. equi strains are similar to that of VapD. It is further shown that sequences encoding putative R. equi Vap-like proteins occur in diverse bacterial species. Finally, the functional implications of the structure are discussed in the light of the unique structural features of VapD and its partial structural similarity to other β-barrel proteins.

An intracellular aliphatic amide degrading inducible amidase produced by Rhodococcus rhodochrous PA-34 was characterized and acrylic acid synthesis from acrylamide was carried out using whole cell amidase. A bioprocess was developed at 50 ml fed batch reaction using 400 mM acrylamide feeding at an interval of 30 min resulted in the production of 4 g acrylic acid with volumetric and catalytic productivity of 80 g/l and 19 g/g/h respectively. The amidase of this organism had molecular weight of 40 kDa and was purified to 8.5 fold with 8% yield. This enzyme was active within the temperature range of 30 to 60 °C, with optimum temperature 45 °C and pH 7.5. The Vmax, Km, and kcat of purified amidase were calculated as 250 U/mg protein, 4.5 mM, and 166 sec-1 for acrylamide. The enzyme showed tolerance to metal chelating agent (EDTA) and was strongly inhibited by heavy metal ions Hg2+, Ag2+, Cu2+ and Co2+. R. rhodochrous PA-34 amidase preferentially hydrolyzed small aliphatic toxic amide such as acrylamide. Thus, the amidase of R. rhodochrous PA-34 is promising biocatalyst for the synthesis of industrially important acids and biodegradation of toxic amides.

Biological oxidation of cyclic ketones normally results in formation of the corresponding dicarboxylic acids, which are further metabolized in the cell. Rhodococcus ruber strain SC1 was isolated from an industrial wastewater bioreactor that was able to utilize cyclododecanone as the sole carbon source. A reverse genetic approach was used to isolate a 10-kb gene cluster containing all genes required for oxidative conversion of cyclododecanone to 1,12-dodecanedioic acid (DDDA). The genes required for cyclododecanone oxidation were only marginally similar to the analogous genes for cyclohexanone oxidation. The biochemical function of the enzymes encoded on the 10-kb gene cluster, the flavin monooxygenase, the lactone hydrolase, the alcohol dehydrogenase, and the aldehyde dehydrogenase, was determined in Escherichia coli based on the ability to convert cyclododecanone. Recombinant E. coli strains grown in the presence of cyclododecanone accumulated lauryl lactone, 12-hydroxylauric acid, and/or DDDA depending on the genes cloned. The cyclododecanone monooxygenase is a type 1 Baeyer-Villiger flavin monooxygenase (FAD as cofactor) and exhibited substrate specificity towards long-chain cyclic ketones (C11 to C15), which is different from the specificity of cyclohexanone monooxygenase favoring short-chain cyclic compounds (C5 to C7).

There is a potential use of oil emulsifying bacteria for the clean-up of oil-contaminated natural environments, and oil transport and storage systems. The control mechanisms of the emulsifying abilities in these bacteria are important in relation to the optimization of such processes. A study was conducted to investigate the physiological criteria for the formation of the emulsifying activity in Rhodococcus sp. strain 094, which forms a hydrophobic cell surface when cultivated on alkanes, permitting oil-associated exponential growth. The ability of this bacteria to produce oil emulsifying agents is clearly inducible by crude oil or a number of single hydrophobic compounds. Before the cells are able to emulsify the oil, they must pass through a relatively short induction period followed by a longer period of synthesis of hydrophobic surface parts, coinciding with cell proliferation. The latter is due to the required coordination with new cell wall synthesis. If the cells are allowed to produce sufficient amounts of hydrophobic cell surface parts, they commence to emulsify the oil efficiently when the hydrophobic factor is released from the cell surface as part of their change to a more hydrophillic state. All the positive inducers were hydrophobic alkyl derivatives, and with one exception, also substrate for growth. Many of the better inducers were alkanes, and would require an alkane oxidizing system to be used by the cells. 14 ref., 2 tabs., 3 figs.

The mechanical properties of Rhodococcus RC291 were measured using force spectroscopy equipped with a bacterial cell probe. Rhodococcal cells in the late growth stage of development were found to have greater adhesion to a silicon oxide surface than those in the early growth stage. This is because there are more extracellular polymeric substances (EPS) that contain nonspecific binding sites available on the cells of late growth stage. It is found that EPS in the late exponential phase are less densely bound but consist of chains able to extend further into their local environment, while the denser EPS at the late stationary phase act more to sheath the cell. Contraction and extension of the EPS could change the density of the binding sites, and therefore affect the magnitude of the adhesion force between the EPS and the silicon oxide surface. By treating rhodococcal EPS as a surface-grafted polyelectrolyte layer and using scaling theory, the interaction between EPS and a solid substrate was modelled for the cell approaching the surface which revealed that EPS possess a large capacity to store charge. Changing the pH of the surrounding medium acts to change the conformation of EPS chains.

Rhodococcus equi is a facultative intracellular opportunistic pathogen of immunocompromised people and a major cause of pneumonia in young horses. An effective live attenuated vaccine would be extremely useful in the prevention of R. equi disease in horses. Toward that end, we have developed an efficient transposon mutagenesis system that makes use of a Himar1 minitransposon delivered by a conditionally replicating plasmid for construction of R. equi mutants. We show that Himar1 transposition in R. equi is random and needs no apparent consensus sequence beyond the required TA dinucleotide. The diversity of the transposon library was demonstrated by the ease with which we were able to screen for auxotrophs and mutants with pigmentation and capsular phenotypes. One of the pigmentation mutants contained an insertion in a gene encoding phytoene desaturase, an enzyme of carotenoid biosynthesis, the pathway necessary for production of the characteristic salmon color of R. equi. We identified an auxotrophic mutant with a transposon insertion in the gene encoding a putative dual-functioning GTP cyclohydrolase II-3,4-dihydroxy-2-butanone-4-phosphate synthase, an enzyme essential for riboflavin biosynthesis. This mutant cannot grow in minimal medium in the absence of riboflavin supplementation. Experimental murine infection studies showed that, in contrast to wild-type R. equi, the riboflavin-requiring mutant is attenuated because it is unable to replicate in vivo. The mutagenesis methodology we have developed will allow the characterization of R. equi virulence mechanisms and the creation of other attenuated strains with vaccine potential.

Two strains of bacteria capable of carrying out the O-methylation of phenolic compounds, one from the gram-positive genus Rhodococcus and one from the gram-negative genus Acinetobacter, were used to examine the O-methylation of phenols carrying fluoro-, chloro-, and bromo-substituents. Zero-order rates of O-methylation were calculated from data for the chloro- and bromophenols; there was no simple relationship between the rate of reaction and the structure of the substrates, and significant differences were observed in the responses of the two test organisms. For the gram-negative strain, the pattern of substitution was as important as the number of substituents. Hexachlorophene was resistant to O-methylation by both strains, and tetrabromobisphenol-A was O-methylated only by the gram-positive strain. It is suggested that in the natural environment, bacterial O-methylation of phenols carrying electron-attracting substituents might be a significant alternative to biodegradation.

Polystyrene, which is one of the most utilized thermoplastics, is highly durable and is considered to be non-biodegradable. Hence, polystyrene waste accumulates in the environment posing an increasing ecological threat. In a previous study we have isolated a biofilm-producing strain (C208) of the actinomycete Rhodococcus ruber that degraded polyethylene films. Formation of biofilm, by C208, improved the biodegradation of polyethylene. Consequently, the present study aimed at monitoring the kinetics of biofilm formation by C208 on polystyrene, determining the physiological activity of the biofilm and analyzing its capacity to degrade polystyrene. Quantification of the biofilm biomass was performed using a modified crystal violet (CV) staining or by monitoring the protein content in the biofilm. When cultured on polystyrene flakes, most of the bacterial cells adhered to the polystyrene surface within few hours, forming a biofilm. The growth of the on polystyrene showed a pattern similar to that of a planktonic culture. Furthermore, the respiration rate, of the biofilm, exhibited a pattern similar to that of the biofilm growth. In contrast, the respiration activity of the planktonic population showed a constant decline with time. Addition of mineral oil (0.005% w/v), but not non-ionic surfactants, increased the biofilm biomass. Extended incubation of the biofilm for up to 8 weeks resulted in a small reduction in the polystyrene weight (0.8% of gravimetric weight loss). This study demonstrates the high affinity of C208 to polystyrene which lead to biofilm formation and, presumably, induced partial biodegradation.

Extracellular polymeric substance (EPS) extracted from Rhodococcus opacus bacterial strain was used as a matrix for calcium carbonate precipitation using the vapour diffusion method. The total exopolymer and water-soluble exopolymer fraction of different concentrations were spread on the mica surface by the spin-coating method. The obtained layers were characterized using the atomic force microscopy measurement and XPS analysis. The effects of polymer concentration, initial pH of calcium chloride solution and precipitation time on the obtained crystals properties were investigated. Raman spectroscopy and scanning electron microscopy were used to characterize the precipitated minerals. It was found that the type of precipitated CaCO3 polymorph and the crystal size depend on the kind of EPS fraction. The obtained results indicates that the water soluble fraction favours vaterite dissolution and calcite growth, whereas the total EPS stabilizes vaterite and this effect is stronger at basic pH. It seems to be due to different contents of the functional group of EPS fractions.

Maintenance of mitotic cell clusters such as meristematic cells depends on their capacity to maintain the balance between cell division and cell differentiation necessary to control organ growth. In the Arabidopsis thaliana root meristem, the antagonistic interaction of two hormones, auxin and cytokinin, regulates this balance by positioning the transition zone, where mitotically active cells lose their capacity to divide and initiate their differentiation programs. In animals, a major regulator of both cell division and cell differentiation is the tumor suppressor protein RETINOBLASTOMA. Here, we show that similarly to its homolog in animal systems, the plant RETINOBLASTOMA-RELATED (RBR) protein regulates the differentiation of meristematic cells at the transition zone by allowing mRNA accumulation of AUXIN RESPONSE FACTOR19 (ARF19), a transcription factor involved in cell differentiation. We show that both RBR and the cytokinin-dependent transcription factor ARABIDOPSIS RESPONSE REGULATOR12 are required to activate the transcription of ARF19, which is involved in promoting cell differentiation and thus root growth.

StCKP1 (Solanum tuberosum cytokinin riboside phosphorylase) catalyses the interconversion of the N9-riboside form of the plant hormone CK (cytokinin), a subset of purines, with its most active free base form. StCKP1 prefers CK to unsubstituted aminopurines. The protein was discovered as a CK-binding activity in extracts of tuberizing potato stolon tips, from which it was isolated by affinity chromatography. The N-terminal amino acid sequence matched the translation product of a set of ESTs, enabling a complete mRNA sequence to be obtained by RACE-PCR. The predicted polypeptide includes a cleavable signal peptide and motifs for purine nucleoside phosphorylase activity. The expressed protein was assayed for purine nucleoside phosphorylase activity against CKs and adenine/adenosine. Isopentenyladenine, trans-zeatin, dihydrozeatin and adenine were converted into ribosides in the presence of ribose 1-phosphate. In the opposite direction, isopentenyladenosine, trans-zeatin riboside, dihydrozeatin riboside and adenosine were converted into their free bases in the presence of Pi. StCKP1 had no detectable ribohydrolase activity. Evidence is presented that StCKP1 is active in tubers as a negative regulator of CKs, prolonging endodormancy by a chill-reversible mechanism.

Maintenance of mitotic cell clusters such as meristematic cells depends on their capacity to maintain the balance between cell division and cell differentiation necessary to control organ growth. In the Arabidopsis thaliana root meristem, the antagonistic interaction of two hormones, auxin and cytokinin, regulates this balance by positioning the transition zone, where mitotically active cells lose their capacity to divide and initiate their differentiation programs. In animals, a major regulator of both cell division and cell differentiation is the tumor suppressor protein RETINOBLASTOMA. Here, we show that similarly to its homolog in animal systems, the plant RETINOBLASTOMA-RELATED (RBR) protein regulates the differentiation of meristematic cells at the transition zone by allowing mRNA accumulation of AUXIN RESPONSE FACTOR19 (ARF19), a transcription factor involved in cell differentiation. We show that both RBR and the cytokinin-dependent transcription factor ARABIDOPSIS RESPONSE REGULATOR12 are required to activate the transcription of ARF19, which is involved in promoting cell differentiation and thus root growth. PMID:24285791

Full Text Available Barley is one of the most important cereal crops grown worldwide. It has numerous applications, but its utility could potentially be extended by genetically manipulating its hormonal balances. To explore some of this potential we identified gene families of cytokinin dehydrogenases (CKX and isopentenyl transferases, enzymes that respectively irreversibly degrade and synthesize cytokinin (CK plant hormones, in the raw sequenced barley genome. We then examined their spatial and temporal expression patterns by immunostaining and qPCR. Two CKX-specific antibodies, anti-HvCKX1 and anti-HvCKX9, predominantly detect proteins in the aleurone layer of maturing grains and leaf vasculature, respectively. In addition, two selected CKX genes were used for stable, Agrobacterium tumefaciens-mediated transformation of the barley cultivar Golden Promise. The results show that constitutive overexpression of CKX causes morphological changes in barley plants and prevents their transition to flowering. In all independent transgenic lines roots proliferated more rapidly and root-to-shoot ratios were higher than in wild-type plants. Only one transgenic line, overexpressing CKX under the control of a promoter from a phosphate transporter gene, which is expressed more strongly in root tissue than in aerial parts, yielded progeny. Analysis of several T1-generation plants indicates that plants tend to compensate for effects of the transgene and restore CK homeostasis later during development. Depleted CK levels during early phases of development are restored by down-regulation of endogenous CKX genes and reinforced de novo biosynthesis of CKs.

Rhodococcus opacus PD630 and Rhodococcus jostii RHA1 are oleaginous bacteria able to synthesize and accumulate triacylglycerols (TAG) in lipid bodies (LB). Highly relevant to the structure of LB is a protein homologous to heparin-binding hemagglutinin (HBHA) (called TadA in rhodococci), which is a virulence factor found in Mycobacterium tuberculosis. HBHA is an adhesin involved in binding to non-phagocytic cells and extrapulmonary dissemination. We observed a conserved synteny of three genes encoding a transcriptional regulator (TR), the HBHA protein and a membrane protein (MP) between TAG-accumulating actinobacteria belonging to Rhodococcus, Mycobacterium, Nocardia and Dietzia genera, among others. A 354 bp-intergenic spacing containing a SigF-binding site was found between hbha and the TR genes in M. tuberculosis, which was absent in genomes of other investigated actinobacteria. Analyses of available "omic" information revealed that TadA and TR were co-induced in rhodococci under TAG-accumulating conditions; whereas in M. tuberculosis and Mycobacterium smegmatis, HBHA and TR were regulated independently under stress conditions occurring during infection. We also found differences in protein lengths, domain content and distribution between HBHA and TadA proteins from mycobacteria and rhodococci, which may explain their different roles in cells. Based on the combination of results obtained in model actinobacteria, we hypothesize that HBHA and TadA proteins originated from a common ancestor, but later suffered a process of functional divergence during evolution. Thus, rhodococcal TadA probably has maintained its original role; whereas HBHA may have evolved as a virulence factor in pathogenic mycobacteria.

Atrazine is a persistent organic pollutant in the environment which affects not only terrestrial and aquatic biota but also human health. Since its removal from the environment is needed, atrazine biodegradation is achieved in the present study using the bacterium Rhodococcus sp. BCH2 isolated from soil, long-term treated with atrazine. The bacterium was capable of degrading about 75 % atrazine in liquid medium having pH 7 under aerobic and dark condition within 7 days. The degradation ability of the bacterium at various temperatures (20-60 °C), pH (range 3-11), carbon (glucose, fructose, sucrose, starch, lactose, and maltose), and nitrogen (ammonium molybdate, sodium nitrate, potassium nitrate, and urea) sources were studied for triumph optimum atrazine degradation. The results indicate that atrazine degradation at higher concentrations (100 ppm) was pH and temperature dependent. However, glucose and potassium nitrate were optimum carbon and nitrogen source, respectively. Atrazine biodegradation analysis was carried out by using high-performance thin-layer chromatography (HPTLC), Fourier transform infrared spectroscopy (FTIR), and liquid chromatography quadrupole time-of-flight (LC/Q-TOF-MS) techniques. LC/Q-TOF-MS analysis revealed formation of various intermediate metabolites including hydroxyatrazine, N-isopropylammelide, deisopropylhydroxyatrazine, deethylatrazine, deisopropylatrazine, and deisopropyldeethylatrazine which was helpful to propose biochemical degradation pathway of atrazine. Furthermore, the toxicological studies of atrazine and its biodegraded metabolites were executed on earthworm Eisenia foetida as a model organism with respect to enzymatic (SOD and Catalase) antioxidant defense mechanism and lipid peroxidation studies. These results suggest innocuous degradation of atrazine by Rhodococcus sp. BCH2 in nontoxic form. Therefore the Rhodococcus sp.BCH2 could prove a valuable source for the eco-friendly biodegradation of atrazine pesticide.

Full Text Available Abstract Background Members of the genus Rhodococcus are frequently found in soil and other natural environments and are highly resistant to stresses common in those environments. The accumulation of storage compounds permits cells to survive and metabolically adapt during fluctuating environmental conditions. The purpose of this study was to perform a genome-wide bioinformatic analysis of key genes encoding metabolism of diverse storage compounds by Rhodococcus jostii RHA1 and to examine its ability to synthesize and accumulate triacylglycerols (TAG, wax esters, polyhydroxyalkanoates (PHA, glycogen and polyphosphate (PolyP. Results We identified in the RHA1 genome: 14 genes encoding putative wax ester synthase/acyl-CoA:diacylglycerol acyltransferase enzymes (WS/DGATs likely involved in TAG and wax esters biosynthesis; a total of 54 genes coding for putative lipase/esterase enzymes possibly involved in TAG and wax ester degradation; 3 sets of genes encoding PHA synthases and PHA depolymerases; 6 genes encoding key enzymes for glycogen metabolism, one gene coding for a putative polyphosphate kinase and 3 putative exopolyphosphatase genes. Where possible, key amino acid residues in the above proteins (generally in active sites, effectors binding sites or substrate binding sites were identified in order to support gene identification. RHA1 cells grown under N-limiting conditions, accumulated TAG as the main storage compounds plus wax esters, PHA (with 3-hydroxybutyrate and 3-hydroxyvalerate monomers, glycogen and PolyP. Rhodococcus members were previously known to accumulate TAG, wax esters, PHAs and polyP, but this is the first report of glycogen accumulation in this genus. Conclusion RHA1 possess key genes to accumulate diverse storage compounds. Under nitrogen-limiting conditions lipids are the principal storage compounds. An extensive capacity to synthesize and metabolize storage compounds appears to contribute versatility to RHA1 in its

Full Text Available The root system is an imperative component of a plant, involved in water and nutrient acquisition from the soil. Any subtle change in the root system may lead to drastic changes in plant productivity. Both auxin and cytokinin are implicated in regulating various root developmental aspects. One of the major signaling cascades facilitating various hormonal and developmental allocations is the Mitogen Activated Protein Kinase (MAPK cascade. Innumerable efforts have been made to unravel the complex nexus involved in rice root development. In spite of a plethora of studies, a comprehensive study aiming to decipher the plausible cross-talk of MAPK signaling module with auxin and cytokinin signaling components in rice is missing. In the present study, extensive phenomics analysis of different stages of rice roots; transcript profiling by qRT-PCR of entire gene family of MAPK, MAPKK and PIN genes; as well as protein level and activity of potential MAPKs was investigated using western and immuno kinase assays both on auxin and cytokinin treatment. The above study led to the identification of various novel rice root specific phenotypic traits by using GiA roots software framework. High expression profile of OsMPK3/6, OsMKK4/5 and OsPIN 1b/9 and their marked transcript level modulation in response to both auxin and cytokinin was observed. Finally, the protein levels and activity assay further substantiated our present findings. Thus, OsMPK3/6-OsMKK4/5 module is elucidated as the putative, key player in auxin-cytokinin interaction augmenting their role by differentially regulating the expression patterns of OsPIN 1b/9 in root development in rice.

One of the most fascinating aspects of plant morphology is the regular geometric arrangement of leaves and flowers, called phyllotaxy. The shoot apical meristem (SAM) determines these patterns, which vary depending on species and developmental stage. Auxin acts as an instructive signal in leaf initiation, and its transport has been implicated in phyllotaxy regulation in Arabidopsis (Arabidopsis thaliana). Altered phyllotactic patterns are observed in a maize (Zea mays) mutant, aberrant phyllotaxy1 (abph1, also known as abphyl1), and ABPH1 encodes a cytokinin-inducible type A response regulator, suggesting that cytokinin signals are also involved in the mechanism by which phyllotactic patterns are established. Therefore, we investigated the interaction between auxin and cytokinin signaling in phyllotaxy. Treatment of maize shoots with a polar auxin transport inhibitor, 1-naphthylphthalamic acid, strongly reduced ABPH1 expression, suggesting that auxin or its polar transport is required for ABPH1 expression. Immunolocalization of the PINFORMED1 (PIN1) polar auxin transporter revealed that PIN1 expression marks leaf primordia in maize, similarly to Arabidopsis. Interestingly, maize PIN1 expression at the incipient leaf primordium was greatly reduced in abph1 mutants. Consistently, auxin levels were reduced in abph1, and the maize PIN1 homolog was induced not only by auxin but also by cytokinin treatments. Our results indicate distinct roles for ABPH1 as a negative regulator of SAM size and a positive regulator of PIN1 expression. These studies highlight a complex interaction between auxin and cytokinin signaling in the specification of phyllotactic patterns and suggest an alternative model for the generation of altered phyllotactic patterns in abph1 mutants. We propose that reduced auxin levels and PIN1 expression in abph1 mutant SAMs delay leaf initiation, contributing to the enlarged SAM and altered phyllotaxy of these mutants.

One of the most fascinating aspects of plant morphology is the regular geometric arrangement of leaves and flowers, called phyllotaxy. The shoot apical meristem (SAM) determines these patterns, which vary depending on species and developmental stage. Auxin acts as an instructive signal in leaf initiation, and its transport has been implicated in phyllotaxy regulation in Arabidopsis (Arabidopsis thaliana). Altered phyllotactic patterns are observed in a maize (Zea mays) mutant, aberrant phyllotaxy1 (abph1, also known as abphyl1), and ABPH1 encodes a cytokinin-inducible type A response regulator, suggesting that cytokinin signals are also involved in the mechanism by which phyllotactic patterns are established. Therefore, we investigated the interaction between auxin and cytokinin signaling in phyllotaxy. Treatment of maize shoots with a polar auxin transport inhibitor, 1-naphthylphthalamic acid, strongly reduced ABPH1 expression, suggesting that auxin or its polar transport is required for ABPH1 expression. Immunolocalization of the PINFORMED1 (PIN1) polar auxin transporter revealed that PIN1 expression marks leaf primordia in maize, similarly to Arabidopsis. Interestingly, maize PIN1 expression at the incipient leaf primordium was greatly reduced in abph1 mutants. Consistently, auxin levels were reduced in abph1, and the maize PIN1 homolog was induced not only by auxin but also by cytokinin treatments. Our results indicate distinct roles for ABPH1 as a negative regulator of SAM size and a positive regulator of PIN1 expression. These studies highlight a complex interaction between auxin and cytokinin signaling in the specification of phyllotactic patterns and suggest an alternative model for the generation of altered phyllotactic patterns in abph1 mutants. We propose that reduced auxin levels and PIN1 expression in abph1 mutant SAMs delay leaf initiation, contributing to the enlarged SAM and altered phyllotaxy of these mutants. PMID:19321707

A cluster of genes encoding a cytochrome P-450 monooxygenase system involved in the utilisation of ethyl tert-butyl ether (ETBE) was cloned in Rhodococcus ruber IFP 2001. This cluster includes ethR, a putative regulator gene of the araC/xylS family; ethA, encoding a ferredoxin reductase; ethB, encoding a cytochrome P-450, ethC, encoding a ferredoxin; and ethD, which is required for the function of the monooxygenase system, but whose exact role is unknown. The ethRABCD cluster is flanked on either side by two identical copies of a class II transposon, which explains that it is lost at high frequency by homologous recombination when the strain is grown under non selective conditions. Two other, highly conserved clusters of eth genes were detected in the ETBE-utilizing strains Rhodococcus zopfii IFP 2005 and Mycobacterium sp. IFP 2009. In all cases, the eth locus is inserted in a different genomic context, suggesting that it may be transferred horizontally between different species and inserted at different sites in the genome. In addition, in R. zopfii IFP 2005, the downstream copy of the transposon carries a 117-bp (base pairs) deletion; in Mycobacterium sp. IFP 2009, the upstream copy is absent and the downstream copy is inserted 2771 bp closer to the ethRABCD cluster. (authors)

Full Text Available Pneumonia caused by Rhodococcus equi is a common cause of disease and death in foals. Although agent and environmental factors contribute to the incidence of this disease, the genetic factors influencing the clinical outcomes of R. equi pneumonia are ill-defined. Here, we performed independent single nucleotide polymorphism (SNP- and copy number variant (CNV-based genome-wide association studies to identify genomic loci associated with R. equi pneumonia in foals. Foals at a large Quarter Horse breeding farm were categorized into 3 groups: 1 foals with R. equi pneumonia (clinical group [N = 43]; 2 foals with ultrasonographic evidence of pulmonary lesions that never developed clinical signs of pneumonia (subclinical group [N = 156]; and, 3 foals without clinical signs or ultrasonographic evidence of pneumonia (unaffected group [N = 49]. From each group, 24 foals were randomly selected and used for independent SNP- and CNV-based genome-wide association studies (GWAS. The SNP-based GWAS identified a region on chromosome 26 that had moderate evidence of association with R. equi pneumonia when comparing clinical and subclinical foals. A joint analysis including all study foals revealed a 3- to 4-fold increase in odds of disease for a homozygous SNP within the associated region when comparing the clinical group with either of the other 2 groups of foals or their combination. The region contains the transient receptor potential cation channel, subfamily M, member 2 (TRPM2 gene, which is involved in neutrophil function. No associations were identified in the CNV-based GWAS. Collectively, these data identify a region on chromosome 26 associated with R. equi pneumonia in foals, providing evidence that genetic factors may indeed contribute to this important disease of foals.

Full Text Available Introduction. Rhodococcus (R equi is an opportunistic, uncommon human pathogen that causes mainly infection in immunocompromised hosts. The disease is usually presented as subacute pneumonia that is mostly cavitary and sometimes bacteremic. Case report. We reported the extremly rare case of a 43-year-old woman with Hodgkin's lymphoma, who developed R. equi pulmonary infection after recieving multiple courses of chemotherapy. Secondary, the patient developed bacteremia, leading to sepsis and dissemination of R. equi infection in many extrapulmonary sites. At addmission the patient was febrile, tachypnoic, tachycardic, hypotensive, with facial edema, splenomegaly, positive meningeal signs, left hemiparesis and paraparesis. Laboratory data included erythrocyte sedimentation rate (ESR > 140 mm/h, C-reactive protein (CRP 143.0 mg/L, red blood cells (RBC 2.14 × 1012/L, whyite blood cells (WBC 2.8 × 109/L, lactate dehydrogenase (LDH 706 U/L, serum albumin 26 g/L, sodium 127 mmol/L and potassium 2.7 mmol/L. Blood culture and culture of sputum and empyema were positive for R. equi. Imaging studies demonstrated a large right cavitary pneumonia and abscess, empyema, pericarditis, mediastinal and intra-abdominal lymphadenopathy, brain and psoas abscesses, osteomyelitis and spondylodiscitis. The patient recovered completely after a 12-month treatment with combinations of parenteral and oral antibiotics (meropenem, vancomycin, teicoplanin, ciprofloxacin, rifampicin, macrolides etc, including drainage of abscesses and empyema. Eight years after completition of the treatment the patient was without recurrence of R. equi infection and lymphoma. Conclusion. Since the eradication od R. equi is very difficult, it is very important to make the diagnosis and initiate appropriate antibiotic therapy as soon as possible.

Full Text Available n order to assess the level of Rhodococcus equi infection in southeast Turkey, 679 sera from healthy foals and adult horses and 78 sera from donkeys were tested by indirect ELISA using a R. equi reference strain (ATCC 33701 as antigen. Eighty (11.7 % sera from horses and 9 (11.5 % sera from donkeys with titres >0.85 were positive. The prevalence of seropositive horses in Sanliurfa Province was higher than in Diyarbakir Province; 56 (13.9 % horses in Sanliurfa Province and 24 (8.7 % horses in Diyarbakir Province were defined as seropositive. In Sanliurfa Province 14.5 % of female (n = 343 and 10.1 % of male (n = 59 horses tested were defined as seropositive, while in Diyarbakir Province more males (11.4 %, n = 114 were seropositive than females (6.7 %, n = 163. Horses 1 to 5 years of age were found to have the highest seropositivity rate in both provinces. A total of 78 sera from donkeys were investigated in Sanliurfa Province, of which 9 (11.5 % were positive by ELISA. Among the 9 positive sera, 6 (12.8 % were from donkeys 1-5 years old and 3 (13.6 % were from donkeys >5years of age. Nopositive sera were found in donkeys less than 1 year old. Five(12.5 % sera of females and 4(10.5 % sera of males tested were positive. These results indicate the existence of R. equi in the horse populations in Sanliurfa and Diyarbakir Provinces. Similar infection rates were found for donkeys in Sanliurfa. This suggests the importance of serological surveys to diagnose R. equi infection in the region and to prevent the zoonotic risk.

Rhodococcus equi is an emerging zoonotic presumably foodborne pathogen. Since the data on the worldwide prevalence of R. equi in meat animals are scarce, the present study aimed to investigate the molecular epidemiology of R. equi in swine, cattle and horse carcasses intended for human consumption in Poland. Totally 1028 lymph node samples were examined. R. equi was isolated from 26.6 % (105/395) swine and 1.3 % (3/234) bovine healthy submaxillary lymph nodes. In horses, R. equi was isolated only from 0.5 % (1/198) samples of middle tracheo-branchiales lymph node while no lymphocentrum retropharyngeum sample was positive (0/198). The purulent lesions were observed only in 0.8 % swine submaxillary lymph nodes samples (3/398) and in two of them R. equi was detected. All bovine and most of swine isolates (98.1 %) were vapB-positive. 87.9 % of swine isolates carried 95-kb type 5 plasmid, 3.7 % type 1 and plasmid types: 4, 7, 10, 11, 21, 31 were carried by a single isolate (0.9 %). All bovine isolates carried VAPB type 26. Single horse isolate was vapA-positive and carried plasmid VAPA 85-kb type I. The prevalence of vapB-positive R. equi in investigated healthy swine intended for human consumption was very high. Not only swine, but also even apparently healthy cattle or horse carcasses should be considered as a potential source of R. equi for humans, especially in countries where undercooked or raw beef or horsemeat is traditionally consumed.

The aim of this study was to determine the MICs of 32 antimicrobial agents for 200 isolates of Rhodococcus equi of animal origin by applying a recently described broth microdilution protocol, and to investigate isolates with distinctly elevated rifampicin MICs for the genetic basis of rifampicin resistance. The study included 200 R. equi isolates, including 160 isolates from horses and 40 isolates from other animal sources, from the USA and Europe. MIC testing of 32 antimicrobial agents or combinations thereof followed a recently published protocol. A novel PCR protocol for the joint amplification of the three rpoB regions in which rifampicin resistance-mediating mutations have been reported was applied to isolates with elevated rifampicin MICs. The amplicons were sequenced and screened for mutations. Susceptibility testing revealed a rather uniform distribution of MICs for most of the antimicrobial agents tested. The lowest MICs were seen for clarithromycin, rifampicin and imipenem. Six isolates (3%) exhibited distinctly higher MICs of rifampicin than the remaining 194 isolates. In five of these six isolates, single bp exchanges, which resulted in the amino acid exchanges Gln513Leu, Asp516Val, His526Asp or Ser531Leu, were detected in the rifampicin resistance-determining region 1 of the rpoB gene, with Gln513Leu representing a novel substitution for R. equi. This study shows the MIC distribution of 32 antimicrobial agents for a large collection of R. equi isolates of animal origin from two continents. Isolates that exhibited distinctly elevated MICs of rifampicin were only rarely detected.

Pneumonia caused by Rhodococcus equi is a common cause of disease and death in foals. Although agent and environmental factors contribute to the incidence of this disease, the genetic factors influencing the clinical outcomes of R. equi pneumonia are ill-defined. Here, we performed independent single nucleotide polymorphism (SNP)- and copy number variant (CNV)-based genome-wide association studies to identify genomic loci associated with R. equi pneumonia in foals. Foals at a large Quarter Horse breeding farm were categorized into 3 groups: 1) foals with R. equi pneumonia (clinical group [N = 43]); 2) foals with ultrasonographic evidence of pulmonary lesions that never developed clinical signs of pneumonia (subclinical group [N = 156]); and, 3) foals without clinical signs or ultrasonographic evidence of pneumonia (unaffected group [N = 49]). From each group, 24 foals were randomly selected and used for independent SNP- and CNV-based genome-wide association studies (GWAS). The SNP-based GWAS identified a region on chromosome 26 that had moderate evidence of association with R. equi pneumonia when comparing clinical and subclinical foals. A joint analysis including all study foals revealed a 3- to 4-fold increase in odds of disease for a homozygous SNP within the associated region when comparing the clinical group with either of the other 2 groups of foals or their combination. The region contains the transient receptor potential cation channel, subfamily M, member 2 (TRPM2) gene, which is involved in neutrophil function. No associations were identified in the CNV-based GWAS. Collectively, these data identify a region on chromosome 26 associated with R. equi pneumonia in foals, providing evidence that genetic factors may indeed contribute to this important disease of foals.

Virulence of the intracellular pathogen Rhodococcus equi depends on a 21.3-kb pathogenicity island located on a conjugative plasmid. To date, the only nonregulatory pathogenicity island-encoded virulence factor identified is the cell envelope-associated VapA protein. Although the pathogenicity islands from porcine and equine R. equi isolates have undergone major rearrangements, the virR operon (virR-icgA-vapH-orf7-virS) is highly conserved in both, suggesting these genes play an important role in pathogenicity. VirR and VirS are transcriptional regulators controlling expression of pathogenicity island genes, including vapA. Here, we show that while vapH and orf7 are dispensable for intracellular growth of R. equi, deletion of icgA, formerly known as orf5, encoding a major facilitator superfamily transport protein, elicited an enhanced growth phenotype in macrophages and a significant reduction in macrophage viability, while extracellular growth in broth remained unaffected. Transcription of virS, located downstream of icgA, and vapA was not affected by the icgA deletion during growth in broth or in macrophages, showing that the enhanced growth phenotype caused by deletion of icgA was not mediated through abnormal transcription of these genes. Transcription of icgA increased 6-fold within 2 h following infection of macrophages and remained significantly higher 48 h postinfection compared to levels at the start of the infection. The major facilitator superfamily transport protein IcgA is the first factor identified in R. equi that negatively affects intracellular replication. Aside from VapA, it is only the second pathogenicity island-encoded structural protein shown to play a direct role in intracellular growth of this pathogenic actinomycete.

To investigate the effect of opsonization of Rhodococcus equi with R. equi-specific antibodies in plasma on bacterial viability and phagocyte activation in a cell culture model of infection. Neutrophils and monocyte-derived macrophages from 6 healthy 1-week-old foals and 1 adult horse. Foal and adult horse phagocytes were incubated with either opsonized or nonopsonized bacteria. Opsonization was achieved by use of plasma containing high or low concentrations of R. equi-specific antibodies. Phagocyte oxidative burst activity was measured by use of flow cytometry, and macrophage tumor necrosis factor (TNF)-α production was measured via an ELISA. Extracellular and intracellular bacterial viability was measured with a novel R. equi-luciferase construct that used a luminometer. Opsonized bacteria increased oxidative burst activity in adult horse phagocytes, and neutrophil activity was dependent on the concentration of specific antibody. Secretion of TNF-α was higher in macrophages infected with opsonized bacteria. Opsonization had no significant effect on bacterial viability in macrophages; however, extracellular bacterial viability was decreased in broth containing plasma with R. equi-specific antibodies, compared with viability in broth alone. The use of plasma enriched with specific antibodies for the opsonization of R. equi increased the activation of phagocytes and decreased bacterial viability in the extracellular space. Although opsonized R. equi increased TNF-α secretion and oxidative burst in macrophages, additional factors may be necessary for effective intracellular bacterial killing. These data have suggested a possible role of plasma antibody in protection of foals from R. equi pneumonia.

Rhodococcus equi is an important bacterial pathogen in foals up to 6 months old, widespread in horse farms all over the world. It was found that only virulent R. equi strains expressing 15-17 kDa virulence-associated protein (VapA) and having large virulence plasmid of 85-90 kb containing vapA gene are pathogenic for horses. To date, 12 plasmid types have been reported in VapA positive strains from horses. There are no data concerning plasmid types of Polish field R. equi strains isolated from horses and horse farm environment. The aim of the study is to determine plasmid profiles of virulent R. equi strains isolated in Poland from dead foals as well as from soil samples taken from horse breeding farms. Plasmid profiles of 10 clinical strains derived from 8 farms and 11 environmental strains from 3 farms, confirmed as virulent by PCR, were compared with 12 reference strains containing the known plasmid size and type. Plasmid DNAs were analysed by digestion with the restriction endonucleases BamHI, EcoRI, EcoT22I, and HindIII for detailed comparison and estimation of plasmid sizes. The results of RFLP analysis revealed that all except one isolates used in the study are classified as VapA 85 kb type I plasmid. One strain harboured VapA 87 kb type I plasmid. This is the first report of plasmid types of Polish field R. equi strains. The results of our preliminary investigations on horse farms located in central and eastern Poland indicate that the virulent R. equi strains thus far isolated from diseased foals and horse farms environment represent a highly uniform plasmid pattern.

Full Text Available Introduction: Biodesulfurization is used as a selective method for lowering the sulfur content of petroleum products. Materials and methods: A sulfur-oxidation bacterial strain named Rhodococcus erythropolis R1 (NCBI GenBank Accession No. GU570564 was used in this study for desulfurization of dibenzothiophene (DBT. Results: The induced culture of strain R1 was able to produce 2-hydroxybiphenyl (2- HBP from DBT followed 4S pathway without further degrading carbon backbone. This process confirmed by gas chromatography (GC analysis. The specific activity of DBT desulfurization by R1 was 45 µM (g dry wt-1 h-1. The addition of Tween 80 as surfactant and glycerol as carbon source determines a 100% rate of DBT-desulfurization during 3 days. The heavy plasmid detected in R1 strain carries dsz genes responsible for biodesulfurization of DBT that was shown by PCR reaction. The mutant strains which had lost this plasmid also had lost desulfurization phenotype. Both mutant and wild strain were sensitive to high concentration of 2-HBP and some antibiotics. Discussion and conclusion: Strain R1 desulfurize DBT through the sulfur-specific degradation pathway or 4S pathway with the selective cleavage of carbon-sulfur (C-S bonds without reducing the energy content. Addition of surfactant enhanced the desulfurization of DBT by increasing its bioavailability and also could improve the growth and desulfurization rate. The location of desulfurization genes was on a heavy plasmid in strain R1. Based on the results of this study, R. erythropolis R1 could serve as a model system for efficient biodesulfurization of petroleum oil without reducing the energy value.

The Rhodococcus jostii RHA1 gene cluster required for γ-resorcylate (GRA) catabolism was characterized. The cluster includes tsdA, tsdB, tsdC, tsdD, tsdR, tsdT, and tsdX, which encode GRA decarboxylase, resorcinol 4-hydroxylase, hydroxyquinol 1,2-dioxygenase, maleylacetate reductase, an IclR-type regulator, a major facilitator superfamily transporter, and a putative hydrolase, respectively. The tsdA gene conferred GRA decarboxylase activity on Escherichia coli. Purified TsdB oxidized NADH in the presence of resorcinol, suggesting that tsdB encodes a unique NADH-specific single-component resorcinol 4-hydroxylase. Mutations in either tsdA or tsdB resulted in growth deficiency on GRA. The tsdC and tsdD genes conferred hydroxyquinol 1,2-dioxygenase and maleylacetate reductase activities, respectively, on E. coli. Inactivation of tsdT significantly retarded the growth of RHA1 on GRA. The growth retardation was partially suppressed under acidic conditions, suggesting the involvement of tsdT in GRA uptake. Reverse transcription-PCR analysis revealed that the tsd genes constitute three transcriptional units, the tsdBADC and tsdTX operons and tsdR. Transcription of the tsdBADC and tsdTX operons was induced during growth on GRA. Inactivation of tsdR derepressed transcription of the tsdBADC and tsdTX operons in the absence of GRA, suggesting that tsd gene transcription is negatively regulated by the tsdR-encoded regulator. Binding of TsdR to the tsdR-tsdB and tsdT-tsdR intergenic regions was inhibited by the addition of GRA, indicating that GRA interacts with TsdR as an effector molecule. PMID:26319878

Acrylonitrile (ACN), a volatile component of the waste generated during the production of acrylamide, also is often associated with aromatic contaminants such as toluene and styrene. Biofiltration, considered an effective technique for the treatment of volatile hydrocarbons, has not been used to treat volatile nitriles. An experimental laboratory-scale trickling bed bioreactor using cells of Rhodococcus rhodochrous DAP 96622 supported on granular activated carbon (GAC) was developed and evaluated to assess the ability of biofiltration to treat ACN. In addition to following the course of treatability of ACN, kinetics of ACN biodegradation during both recycle batch and open modes of operation by immobilized and free cells were evaluated. For fed-batch mode bioreactor with immobilized cells, almost complete ACN removal (>95%) was achieved at a flow rate of 0.1 microl/min ACN and 0.8 microl/min toluene (TOL) (for comparative purposes this is equivalent to 6.9 mg l(-1) h(-1) ACN and 83.52 mg l(-1) h(-1) TOL). In a single-pass mode bioreactor with immobilized cells, at ACN inlet loads of 100-200 mg l(-1) h(-1) and TOL inlet load of approximately 400 mg l(-1) h(-1), with empty bed retention time (EBRT) of 8 min, ACN removal efficiency was approximately 90%. The three-dimensional structure and characteristics of the biofilm were investigated using confocal scanning laser microscopy (CSLM). CLSM images revealed a robust and heterogeneous biofilm, with microcolonies interspersed with voids and channels. Analysis of the precise measurement of biofilm characteristics using COMSTAT agreed with the assumption that both biomass and biofilm thickness increased along the carbon column depth.

Full Text Available The aim of this study was to determine the influence of meta-topolin (mT and 6-benzyl-aminopurine (BAP on the hydrogen peroxide (H2O2 level and antioxidant enzymes activities in relation to the shoot formation and senescence process in Pelargonium hortorum cultivars, which differ in their susceptibility to leaf yellowing under in vitro conditions. In an early senescing cultivar ‘Grand Prix’, the addition of an aromatic cytokinin mT to abscisic acid (ABA-enriched Murashige and Skoog (MS basal medium more efficiently inhibited leaf yellowing than BAP. In both genotypes, meta-topolin was also the most effective in shoot formation. It was found that Pelargonium species varying in their susceptibility to senescence differ in H2O2 production and antioxidant enzymes activities. Generally, meta-topolin more effectively enhanced H2O2 production and POD activity than BAP and control medium, but its effect depended on genotype. The highest H2O2 production stimulated by mT was observed on day 5 of subculture in late senescing cv. ‘Bergpalais’. In both geranium genotypes, superoxide dismutase (SOD and catalase (CAT levels were highest at the beginning of the subculture period, during the initiation of shoot formation. SOD showed the highest activity on day 5 of subculture on the medium without cytokinin and generally being higher in cv. ‘Bergpalais’ than in cv. ‘Grand Prix’. CAT activity was positively regulated by both cytokinins. POD activity was most effectively enhanced by mT, but on different days of subculture - on the 2nd day of subculture in cv. ‘Bergpalais’ and on the 22nd day of subculture in cv. ‘Grand Prix’. The enhanced activity of POD in the presence of mT, 4-fold higher than on control medium, at the end of subculture in P. hortorum ‘Grand Prix’ coincided with the inhibition of leaf senescence.

Full Text Available Stay-green, a key trait of wheat, can not only increase the yield of wheat but also its resistance to heat stress during active photosynthesis. Cytokinins are the most potent general coordinator between the stay-green trait and senescence. The objectives of the present study were to identify and assess the effects of cytokinins on the photosynthetic organ and heat resistance in wheat. Two winter wheat cultivars, Wennong 6 (a stay-green cultivar and Jimai 20 (a control cultivar, were subjected to heat stress treatment from 1 to 5 days after anthesis (DAA. The two cultivars were sprayed daily with 10 mg L-1 of 6-benzylaminopurine (6-BA between 1 and 3 DAA under ambient and elevated temperature conditions. We found that the heat stress significantly decreased the number of kernels per spike and the grain yield (P < 0.05. Heat stress also decreased the zeatin riboside (ZR content, but increased the gibberellin (GA3, indole-3-acetic acid (IAA, and abscisic acid (ABA contents at 3 to 15 DAA. Application of 6-BA significantly (P < 0.05 increased the grain-filling rate, endosperm cell division rate, endosperm cell number, and 1,000-grain weight under heated condition. 6-BA application increased ZR and IAA contents at 3 to 28 DAA, but decreased GA3 and ABA contents. The contents of ZR, ABA, and IAA in kernels were positively and significantly correlated with the grain-filling rate (P < 0.05, whereas GA3 was counter-productive at 3 to 15 DAA. These results suggest that the decrease in grain yield under heat stress was due to a lower ZR content and a higher GA3 content compared to that at elevated temperature during the early development of the kernels, which resulted in less kernel number and lower grain-filling rate. The results also provide essential information for further utilization of the cytokinin substances in the cultivation of heat-resistant wheat.

This paper reports the biochemical characterization of a purified and reconstituted two-component 3-ketosteroid 9 alpha-hydroxylase (KSH). KSH of Rhodococcus rhodochrous DSM 43269, consisting of a ferredoxin reductase (KshB) and a terminal oxygenase (KshA), was heterologously expressed in

3-Hydroxybenzoate 6-hydroxylase (3HB6H) from Rhodococcus jostii RHA1 is an NADH-specific flavoprotein monooxygenase that catalyzes the para-hydroxylation of 3-hydroxybenzoate (3HB) to form 2,5-dihydroxybenzoate (2,5-DHB). Based on results from stopped-flow spectrophotometry, the reduced enzyme-3HB c

In root exudates of alfalfa, wheat and radish we have found 12 amino acids. As well as 5 organic acids were identified. In the variant with exudates of wheat as the sole source of carbon and energy maximum titer of microorganism Rhodococcus erythropolis RCM Ac-2017D were observed

Full Text Available In root exudates of alfalfa, wheat and radish we have found 12 amino acids. As well as 5 organic acids were identified. In the variant with exudates of wheat as the sole source of carbon and energy maximum titer of microorganism Rhodococcus erythropolis RCM Ac-2017D were observed

Ethyl tert-butyl ether (ETBE) enrichment was obtained by adding contaminated groundwater to a mineral medium containing ETBE as the sole carbon and energy source. ETBE was completely degraded to biomass and CO2 with a transient production of tert-butanol (TBA) and a final biomass yield of 0.37 ± 0.08 mg biomass (dry weight).mg(-1) ETBE. Two bacterial strains, IFP 2042 and IFP 2049, were isolated from the enrichment, and their 16S rRNA genes (rrs) were similar to Rhodococcus sp. (99 % similarity to Rhodococcus erythropolis) and Bradyrhizobium sp. (99 % similarity to Bradyrhizobium japonicum), respectively. Rhodococcus sp. IFP 2042 degraded ETBE to TBA, and Bradyrhizobium sp. IFP 2049 degraded TBA to biomass and CO2. A mixed culture of IFP 2042 and IFP 2049 degraded ETBE to CO2 with a biomass yield similar to the original ETBE enrichment (0.31 ± 0.02 mg biomass.mg(-1) ETBE). Among the genes previously described to be involved in ETBE, MTBE, and TBA degradation, only alkB was detected in Rhodococcus sp. IFP 2042 by PCR, and none were detected in Bradyrhizobium sp. IFP 2049.

Rhodococcus sp. strain YU6 was isolated from soil for the ability to grow on o-xylene as the sole carbon and energy source. Unlike most other o-xylene-degrading bacteria, YU6 is able to grow on p-xylene. Numerous growth substrate range experiments, in addition to the ring-cleavage enzyme assay data, suggest that YU6 initially metabolizes o- and p-xylene by direct aromatic ring oxidation. This leads to the formation of dimethylcatechols, which was further degraded largely through meta-cleavage pathway. The gene encoding meta-cleavage dioxygenase enzyme was PCR cloned from genomic YU6 DNA using previously known gene sequence data from the o-xylene-degrading Rhodococcus sp. strain DK17. Subsequent sequencing of the 918-bp PCR product revealed a 98% identity to the gene, encoding methylcatechol 2,3-dioxygenase from DK17. PFGE analysis followed by Southern hybridization with the catechol 2,3-dioxygenase gene demonstrated that the gene is located on an approximately 560-kb megaplasmid, designated pJYJ1.

The goal of this study was to compare the degradation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by three Rhodococcus strains under anaerobic, microaerophilic (RDX as the sole nitrogen source. RDX and RDX metabolite concentrations were measured over time. Under microaerophilic conditions, the bacteria degraded RDX, albeit about 60-fold slower than under fully aerobic conditions. Only the breakdown product, 4-nitro-2,4-diazabutanal (NDAB) accumulated to measurable concentrations under microaerophilic conditions. RDX degraded quickly under both aerated and static aerobic conditions (DO allowed to drop below 1 mg l(-1)) with the accumulation of both NDAB and methylenedinitramine (MEDINA). No RDX degradation was observed under strict anaerobic conditions. The Rhodococcus strains did not degrade RDX under strict anaerobic conditions, while slow degradation was observed under microaerophilic conditions. The RDX metabolite NDAB was detected under both microaerophilic and aerobic conditions, while MEDINA was detected only under aerobic conditions. IMPACT AND SIGNIFICANCE OF THE STUDY: This work confirmed the production of MEDINA under aerobic conditions, which has not been previously associated with aerobic RDX degradation by these organisms. More importantly, it demonstrated that aerobic rhodococci are able to degrade RDX under a broader range of oxygen concentrations than previously reported.

The identification of fastidious aerobic Actinomycetes such as Gordonia, Rhodococcus, and Tsukamurella has remained a challenge leading to clinically significant misclassifications. This study is intended to examine the feasibility of partial 5'-end 16S rRNA gene sequencing for the identification of Gordonia, Rhodococcus, and Tsukamurella, and defined potential reference sequences for species from each of these genera. The 16S rRNA gene sequence based identification algorithm for species identification was used and enhanced by aligning test sequences with reference sequences from the List of Prokaryotic Names with Standing in Nomenclature. Conventional PCR based 16S rRNA gene sequencing and the alignment of the isolate 16S rRNA gene sequence with reference sequences accurately identified 100% of clinical strains of aerobic Actinomycetes. While partial 16S rRNA gene sequences of reference type strains matched with the 16S rRNA gene sequences of 19 isolates in our data set, another 13 strains demonstrated a degree of polymorphism with a 1-4 bp difference in the regions of difference. 5'-end 606 bp 16S rRNA gene sequencing, coupled with the assignment of well defined reference sequences to clinically relevant species of bacteria, can be a useful strategy for improving the identification of clinically relevant aerobic Actinomycetes.

In the present work, statistical experimental methodology was used to enhance the production of amidase from Rhodococcus erythropolis MTCC 1526. R. erythropolis MTCC 1526 was selected through screening of seven strains of Rhodococcus species. The Placket-Burman screening experiments suggested that sorbitol as carbon source, yeast extract and meat peptone as nitrogen sources, and acetamide as amidase inducer are the most influential media components. The concentrations of these four media components were optimised using a face-centred design of response surface methodology (RSM). The optimum medium composition for amidase production was found to contain sorbitol (5 g/L), yeast extract (4 g/L), meat peptone (2.5 g/L), and acetamide (12.25 mM). Amidase activities before and after optimisation were 157.85 units/g dry cells and 1,086.57 units/g dry cells, respectively. Thus, use of RSM increased production of amidase from R. erythropolis MTCC 1526 by 6.88-fold.

Full Text Available The aim of this study was to determine the activity of cytokinin dehydrogenase (CKX and to measure other biochemical components in the primary leaves and radicles of castor seedlings (BRS Energia in the initial phase of growth. The crude protein extract obtained after a 1-h extraction from the root tissues of seedlings showed no detectable CKX enzymatic activity when incubated with the substrate isopentenyl adenine for 1 h. However, after precipitation with ammonium sulfate at 70% saturation, the pellet showed CKX activity. The peroxidase enzyme activity was higher in the leaves than in the radicles. The total and reducing sugar content was 1.5 times higher in the leaves than in the radicles. The amino acid and protein contents were 6.4 and 9.2 times higher in the leaves than in the radicles, respectively.

Full Text Available Cytokinin oxidase/dehydrogenase (CKX; EC.1.5.99.12 regulates cytokinin (CK level in plants and plays an essential role in CK regulatory processes. CKX proteins are encoded by a small gene family with a varying number of members in different plants. In spite of their physiological importance, systematic analyses of SiCKX genes in foxtail millet have not yet been examined. In this paper, we report the genome wide isolation and characterization of SiCKXs using bioinformatic methods. A total of 11 members of the family were identified in the foxtail millet genome. SiCKX genes were distributed in seven chromosomes (chromosome 1, 3, 4, 5, 6, 7, and 11. The coding sequences of all the SiCKX genes were disrupted by introns, with numbers varying from one to four. These genes expanded in the genome mainly due to segmental duplication events. Multiple alignment and motif display results showed that all SiCKX proteins share FAD- and CK-binding domains. Putative cis-elements involved in Ca2 +-response, abiotic stress response, light and circadian rhythm regulation, disease resistance and seed development were present in the promoters of SiCKX genes. Expression data mining suggested that SiCKX genes have diverse expression patterns. Real-time PCR analysis indicated that all 11 SiCKX genes were up-regulated in embryos under 6-BA treatment, and some were NaCl or PEG inducible. Collectively, these results provide molecular insights into CKX research in plants.

Immune adult horses have CD8+ cytotoxic T lymphocytes (CTLs) that recognize and lyse Rhodococcus equi-infected cells in an equine lymphocyte alloantigen (ELA)-A [classical major histocompatibility complex (MHC) class I]-unrestricted fashion. As protein antigens are MHC class I-restricted, the lack of restriction suggests that the bacterial antigens being recognized by the host are not proteins. The goals of this study were to test the hypothesis that these CTLs recognize unique R. equi cell-wall lipids related to mycobacterial lipids. Initial experiments showed that treatment of soluble R. equi antigen with broadly reactive proteases did not significantly diminish the ability of the antigen to stimulate R. equi-specific CTLs. R. equi-specific CTLs were also shown to lyse target cells (equine macrophages) pulsed with an R. equi lipid extract. Analysis of the R. equi lipid by TLC and MS (MALDI-TOF and ES) indicated that the extracted antigen consisted of three primary fractions: trehalose monomycolate (TMM), trehalose dimycolate (TDM) and cardiolipin (CL). ELA-A-mismatched cells pulsed with purified TMM and CL, but not the TDM fraction, were recognized and lysed by R. equi-specific CTLs. Because of their role in immune clearance and pathogenesis, transcription of the cytokines gamma interferon (IFN-γ) and interleukin-4 (IL-4) was also measured in response to R. equi lipids by using real-time PCR; elevated IFN-γ, but not IL-4, was associated with host clearance of the bacteria. The whole-cell R. equi lipid and all three R. equi lipid fractions resulted in marked increases in IFN-γ transcription, but no increase in IL-4 transcription. Together, these data support the hypothesis that immune recognition of unique lipids in the bacterial cell wall is an important component of the protective immune response to R. equi. The results also identify potential lipid antigens not previously shown to be recognized by CTLs in an important, naturally occurring actinomycete

Background Rhodococcus equi (R.equi) infection commonly occurs in grazing areas,especially in patients with AIDS or with T-lymphocyte immuno-deficiencies. Literature reviews revealed that cases radiologically and pathologically diagnosed of AIDS complicated by R. equi infection are rare. This study aimed to investigate the imaging features and pathological basis of AIDS complicated by pulmonary R. equi infection.Methods A total of 13 cases of AIDS complicated by pulmonary R. equi infection were retrospectively analyzed based on their imaging,bacterial culture and pathological data,including 10 cases by chest CT scanning and X-ray radiology and 3 cases by only X-ray radiology. All 13 cases were definitely diagnosed by bacterial culture,including one by CT-guided pulmonary puncture with following H&E staining and periodic acid-Schiff (PAS) staining for diagnostic biopsy and another one by bronchial biopsy with following H&E staining and PAS staining for pathological diagnosis. The imaging findings and the pathological findings of AIDS complicated by pulmonary R. equi infection were compared and evaluated.Results Totally 9 subjects (70%) had radiological demonstrations of central ball liked high density shadows in unilateral pulmonary hilus areas;10 (77%),cavities and liquefied levels;3 (23%),pleural effussion. The foci were found in pulmonary inner zone in 10 subjects (77%) and in pulmonary outer zone in one subject (7%). The pathological findings included intra-alveolar hemorrhage,lymphocyte infiltration and granulation tissue proliferation,which were in line with the pathological process of necrotic pneumonia. After 8-month follow-up of anti-R. equi therapy of these 13 cases,9 cases had obviously decreased or shrunk pulmonary cavities,one died,one missed follow-up,one completely absorbed foci and one did not receive reexaminations.Conclusions The radiological demonstrations of AIDS complicated by pulmonary R. equiinfection are central ball liked high density areas

Full Text Available The humoral immune response in ‘Brasileiro de Hipismo’ (BH breed and Breton mares was compared after using the Rhodococcus equi vaccine, and the effect of maternal immunoprophylaxis on antibody transfer to newborn foals through the colostrum was evaluated. Blood samples were obtained from 16 pregnant mares vaccinated against R. equi, 16 foals (offspring from vaccinated mares, 8 unvaccinated pregnant mares and 8 foals (offspring from control mares. R equi serum antibody titers were determined by enzyme-linked immunosorbant assay (ELISA after the immunization of pregnant mares using two different antigens, APTX and the commercial vaccine. There was no difference in antibody production between the two breeds. Significant increase in R. equi antibody titers was observed in mares after vaccination (p<0.01, reaching a peak at foaling. Afterward, titers tended to decrease for up to 60 days after birth (dab and then remained constant until 150 dab. Significant antibody transfer to the vaccinated mares newborn foal occurred through the colostrum. A slight reduction in antibody titer was observed at 60 dab, after which titers remained constant for up to 150 dab. The commercial antigen detected significantly higher antibody titers than did APTX (p<0.01.

The activity of the phytohormone cytokinin depends on a complex interplay of factors such as its metabolism, transport, stability, and cellular/tissue localization. O-glucosides of zeatin-type cytokinins are postulated to be storage and/or transport forms, and are readily deglucosylated. Transgenic tobacco (Nicotiana tabacum L. cv. Petit Havana SR1) plants were constructed over-expressing Zm-p60.1, a maize beta-glucosidase capable of releasing active cytokinins from O- and N3-glucosides, to analyse its potential to perturb zeatin metabolism in planta. Zm-p60.1 in chloroplasts isolated from transgenic leaves has an apparent K(m) more than 10-fold lower than the purified enzyme in vitro. Adult transgenic plants grown in the absence of exogenous zeatin were morphologically indistinguishable from the wild type although differences in phytohormone levels were observed. When grown on medium containing zeatin, inhibition of root elongation was apparent in all seedlings 14 d after sowing (DAS). Between 14 and 21 DAS, the transgenic seedlings accumulated fresh weight leading later (28-32 DAS) to ectopic growths at the base of the hypocotyl. The development of ectopic structures correlated with the presence of the enzyme as demonstrated by histochemical staining. Cytokinin quantification showed that transgenic seedlings grown on medium containing zeatin accumulate active metabolites like zeatin riboside and zeatin riboside phosphate and this might lead to the observed changes. The presence of the enzyme around the base of the hypocotyl and later, in the ectopic structures themselves, suggests that the development of these structures is due to the perturbance in zeatin metabolism caused by the ectopic presence of Zm-p60.1.

The oncogenic 6b gene of Agrobacterium tumefaciens induces a number of morphological and metabolic alterations in plants. Although molecular functions associated with the 6b genes have been proposed, including auxin transport, sugar transport, transcriptional regulation, and miRNA metabolism, so far an unequivocal conclusion has not been obtained. We investigated the association between auxin accumulation and tumor development of the tobacco seedlings expressing the AK-6b gene under the control of the dexamethasone-inducible promoter. Indole-3-acetic acid (IAA) localization was examined by immunochemical staining with monoclonal antibody against IAA and by histochemical analysis using the IAA-specific induced construct, DR5::GUS (β-glucuronidase). Both procedures indicated that IAA preferentially accumulated in the tumorous protrusions as well as in newly developing vascular bundles in the tumors. Furthermore, true leaves also showed abaxial IAA localization, leading to altered leaves in which the adaxial and abaxial identities were no longer evident. Co-localization of cytokinin and auxin in the abaxial tumors was verified by immunochemical staining with an antibody against cytokinin. Treatment of AK-6b-seedlings with N-1-naphthylphthalamic acid, an inhibitor of polar auxin transport, promoted the morphological severity of phenotypes, whereas 1-naphthoxyacetic acid, a specific auxin influx carrier inhibitor, induced tumor regression on cotyledons and new tumorous proliferations on hypocotyls. Prominent accumulation of both auxin and cytokinin was observed in both regressed and newly developing tumors. We suggest from these results that modulation of auxin/cytokinin localization as a result of AK-6b gene expression is responsible for the tumorous proliferation.

Full Text Available A rodococose é uma doença de distribuição mundial causada pelo Rhodococcus equi, responsável por taxas elevadas de mortalidade e grandes perdas econômicas relacionadas à pneumonia grave em potros com menos de seis meses de idade. Essa revisão inclui a etiologia, epidemiologia e patogenia da doença com atenção especial à proteína de superfície VapA, seu principal determinante de virulência. As principais manifestações clínicas são apresentadas, bem como os métodos diagnósticos e as suas aplicações, incluindo as novas estratégias em desenvolvimento. Da mesma maneira, as medidas terapêuticas mais utilizadas são também discutidas, abordando principalmente o uso de antibióticos capazes de penetrar nas formações abscedantes.Rodococosis is a disease that has a worldwide distribution caused by Rhodococcus equi. In foals under six months high mortality and great econimic losses are related to this bacterial pneumonia. This review includes the ethiology, epidemiology and pathogenesis of the disease with focus on the role of VapA, a surface protein, as the major determinant of virulence. The clinical manifestations are reviewed and diagnostic methods and their applications are commented, including new strategies that are still being developed. Likewise, the most common clinical therapies are discussed specially those using antibiotics that are known to penetrate in abcesses.

The ability of a Rhodococcus strain to produce surface-active agents from residual sunflower frying oil (RSFO) has been screened in batch cultures. During cultivation with RSFO at the concentration 3% (vol/vol), the strain has synthesized extra-cellular compounds which increase the E{sub 24} emulsion index of the culture medium up to 63%. In their crude form, these substances lower the surface tension of water until 31.9 mN m{sup -1}. The exponential growth with RSFO as the sole carbon source has developed at a specific growth rate {mu} = 0.55 d{sup -1}. The critical micelle concentration of the crude product reached the value 287 mg L{sup -1} ({gamma}CMC = 31.9 mN m{sup -1}). After methyl-esterification, the lipid fraction of bio-surfactants has been analyzed by GC-MS in EI, which reveals the presence of fatty acid methyl esters. The microorganism was also cultivated with the diesel oil as the sole carbon source at the concentration 1% (vol/vol): the active growth phase has developed at rate = 0.02 d{sup -1}, without production of emulsifying substance: the microorganism seems to develop different modes of substrate uptake, according to the nature of the carbon source. The potential use of surface-active agents synthesized on RSFO by Rhodococcus erythropolis 16 LM.USTHB is in the oil industry with minimum purity specification, so that crude preparation could be used, at low cost, in clean-up of hydrocarbons contaminated sites and for enhanced oil recovery. (authors)

The two-component signaling system--the major signaling pathway of bacteria--is found among higher eukaryotes only in plants, where it regulates diverse processes, such as the signaling of the phytohormone cytokinin. Cytokinin is perceived by a hybrid histidine (His) kinase receptor, and the signal is transduced by a multistep phosphorelay system of His phosphotransfer proteins and different classes of response regulators (RRs). To shed light on the origin and evolution of the two-component signaling system members in plants, we conducted a comprehensive domain-based phylogenetic study across the relevant kingdoms, including Charophyceae algae, the group of green algae giving rise to land plants. Surprisingly, we identified a subfamily of cytokinin receptors with members only from the early diverging land plants Marchantia polymorpha and Physcomitrella patens and then experimentally characterized two members of this subfamily. His phosphotransfer proteins of Charophyceae seemed to be more closely related to land plants than to other groups of green algae. Farther down the signaling pathway, the type-B RRs were found across all plant clades, but many members lack either the canonical Asp residue or the DNA binding domain. In contrast, the type-A RRs seemed to be limited to land plants. Finally, the analysis provided hints that one additional group of RRs, the type-C RRs, might be degenerated receptors and thus, of a different evolutionary origin than bona fide RRs.

Full Text Available Ustilago maydis is the causative agent of common smut of corn. Early studies noted its ability to synthesize phytohormones and, more recently these growth promoting substances were confirmed as cytokinins (CKs. Cytokinins comprise a group of phytohormones commonly associated with actively dividing tissues. Lab analyses identified variation in virulence between U. maydis dikaryon and solopathogen infections of corn cob tissue. Samples from infected cob tissue were taken at sequential time points post infection and biochemical profiling was performed using high performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI MS/MS. This hormone profiling revealed that there were altered levels of ABA and major CKs, with a marked reduction in CK glucosides, increases in methylthiol CKs and a particularly dramatic increase in cisZ CK forms, in U. maydis infected tissue. These changes were more pronounced in the more virulent dikaryon relative to the solopathogenic strain suggesting a role for cytokinins in moderating virulence during biotrophic infection. These findings highlight the fact that U. maydis does not simply mimic a fertilized seed but instead reprograms the host tissue. Results underscore the suitability of the Ustilago maydis- Zea mays model as a basis for investigating the control of phytohormone dynamics during biotrophic infection of plants.

Ustilago maydis is the causative agent of common smut of corn. Early studies noted its ability to synthesize phytohormones and, more recently these growth promoting substances were confirmed as cytokinins (CKs). Cytokinins comprise a group of phytohormones commonly associated with actively dividing tissues. Lab analyses identified variation in virulence between U. maydis dikaryon and solopathogen infections of corn cob tissue. Samples from infected cob tissue were taken at sequential time points post infection and biochemical profiling was performed using high performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI MS/MS). This hormone profiling revealed that there were altered levels of ABA and major CKs, with a marked reduction in CK glucosides, increases in methylthiol CKs and a particularly dramatic increase in cisZ CK forms, in U. maydis infected tissue. These changes were more pronounced in the more virulent dikaryon relative to the solopathogenic strain suggesting a role for cytokinins in moderating virulence during biotrophic infection. These findings highlight the fact that U. maydis does not simply mimic a fertilized seed but instead reprograms the host tissue. Results underscore the suitability of the Ustilago maydis- Zea mays model as a basis for investigating the control of phytohormone dynamics during biotrophic infection of plants.

Virulence of Rhodococcus equi strains from 20 humans in Brazil was investigated by using a polymerase chain reaction to characterize isolates as virulent (VapA), intermediately virulent (VapB), and avirulent. Nine isolates were obtained from human immunodeficiency virus (HIV)–positive patients, six from HIV-negative patients, and five from patients of unknown status. Five isolates were VapB positive, four were VapA positive, and eleven were avirulent. Among the nine isolates from HIV-positive patients, five contained VapB plasmids and two contained VapA plasmids. Five VapB-positive isolates had the type 8 virulence plasmid. Eleven of the patients had a history of contact with livestock and/or a farm environment, and none had contact with pigs. PMID:21896813

The alkane hydroxylase systems of two Rhodococcus strains (NRRL B-16531 and Q15, isolated from different geographical locations) were characterized. Both organisms contained at least four alkane monooxygenase gene homologs (alkB1, alkB2, alkB3, and alkB4). In both strains, the alkB1 and alkB2 homologs were part of alk gene clusters, each encoding two rubredoxins (rubA1 and rubA2; rubA3 and rubA4), a putative TetR transcriptional regulatory protein (alkU1; alkU2), and, in the alkB1 cluster, a ...

Novel isolates of Achromobacter xylosoxidans, Tsukamurella wratislaviensis and a Rhodococcus sp. are described. These grew with short-chain alkanesulfonates as their sole source of carbon and energy. T. wratislaviensis strain SB2 grew well with C(3)-C(6) linear alkanesulfonates, isethionate and taurine, Rhodococcus sp. strain CB1 used C(3)-C(10) linear alkanesulfonates, taurine and cysteate, but neither strain grew with ethanesulfonate. In contrast, A. xylosoxidans strain AE4 grew well with ethanesulfonate, making it the first bacterium to be described which can grow with this compound. It also grew with unsubstituted C(3)-C(5) alkanesulfonates and isethionate. Hydrolysis was excluded as a mechanism for alkanesulfonate metabolism in these strains; and evidence is given for a diversity of uptake and desulfonatase systems. We provide evidence for an initial monooxygenase-dependent desulfonation in the metabolism of ethanesulfonate and propanesulfonate by A. xylosoxidans strain AE4.

Rhodococcus opacus R7 is a Gram-positive bacterium isolated from a polycyclic aromatic hydrocarbon contaminated soil for its versatile metabolism; indeed the strain is able to grow on naphthalene, o-xylene, and several long- and medium-chain n-alkanes. In this work we determined the degradation of n-alkanes in Rhodococcus opacus R7 in presence of n-dodecane (C12), n-hexadecane (C16), n-eicosane (C20), n-tetracosane (C24) and the metabolic pathway in presence of C12. The consumption rate of C12 was 88%, of C16 was 69%, of C20 was 51% and of C24 it was 78%. The decrement of the degradation rate seems to be correlated to the length of the aliphatic chain of these hydrocarbons. On the basis of the metabolic intermediates determined by the R7 growth on C12, our data indicated that R. opacus R7 metabolizes medium-chain n-alkanes by the primary alcohol formation. This represents a difference in comparison with other Rhodococcus strains, in which a mixture of the two alcohols was observed. By GC-MSD analysis we also identified the monocarboxylic acid, confirming the terminal oxidation. Moreover, the alkB gene cluster from R. opacus R7 was isolated and its involvement in the n-alkane degradation system was investigated by the cloning of this genomic region into a shuttle-vector E. coli-Rhodococcus to evaluate the alkane hydroxylase activity. Our results showed an increased biodegradation of C12 in the recombinant strain R. erythropolis AP (pTipQT1-alkR7) in comparison with the wild type strain R. erythropolis AP. These data supported the involvement of the alkB gene cluster in the n-alkane degradation in the R7 strain.

Genes homologous to enterococcal glycopeptide resistance genes vanA and vanB were found in glycopeptide-resistant Paenibacillus and Rhodococcus strains from soil. The putative D-Ala:D-Lac ligase genes in Paenibacillus thiaminolyticus PT-2B1 and Paenibacillus apiarius PA-B2B were closely related...... to vanA (92 and 87%) and flanked by genes homologous to vanH and vanX in vanA operons....

红球菌属是有机污染物降解的重要微生物之一.由于红球菌能够适应各种各样的底物环境,具有极强的有机溶剂耐受性和很宽的降解谱,同时它们还能通过产生表面活性剂和改变细胞表面组成结构来提升自身对于疏水性环境的适应能力,因此,红球菌在石油污染物降解及石油污染的生物修复等领域有着极其重要的应用价值.文章基于近年来在红球菌降解石油烃方面的研究进展,从红球菌适应疏水性环境的机制、石油烃中烷烃、环烷烃、芳香烃的降解途径等几个方面进行综述,同时对今后研究的方向进行了展望.%Rhodococcus as a sort of microbial for organic compounds biodegradation with strong organic solvent tolerance and wide degradation spectrum, can adapt to various substrates, as well as promoting its adaption to hydrophobic surroundings by producing surfactants or changing cell surface composition and structure.Due to the characteristics, Rhodococcus acts extremely important roles in oil pollution bioremediation.Based on progress in petroleum hydrocarbon biodegradation by Rhodococcus, the adaption mechanism to hydrophobic environment, the degradation process of alkane, cycloparafin hydrocarbon and aromatics were reviewed.The prospect related to Rhodococcus in bioremediation in future was also proposed.

Full Text Available Triatomines are insects of great importance for public health because they transmit the parasite Trypano-soma cruzi, etiological agent of Chagas disease. These insects harbour intestinal symbiotic flora from theRhodococcus genus, which provide the nutrients lacking in the insect diet, nutrients which are essentialfor normal insect development and growth. Study of these bacteria is of special interest due to thepossibility of designing paratransgenesis control strategies as well as for implications that interactionsbetween these bacteria and parasites can develop inside triatomine insects. Taking into account thedifficulty in identifying bacteria from the Rhodococcus genus by traditional methods, in this work apolymerase chain reaction (PCR followed by a restriction fragment length polymorphism analysis wasstandardized to identify Rhodococcus rhodnii isolated from Rhodnius prolixus. Gene coding for bacterial16 S rRNA was amplified and obtained products were sequenced and digested with several restrictionenzymes. Results show a total correlation between number and size of the restriction fragments obtainedin vitro and in silico as well. Therefore, applicability of this PCR-RFLP assay for R. rhodnii identificationhas been demonstrated.

Arabidopsis (Arabidopsis thaliana) SPINDLY (SPY) is a putative serine and threonine O-linked N-acetylglucosamine transferase (OGT). While SPY has been shown to suppress gibberellin signaling and to promote cytokinin (CK) responses, its catalytic OGT activity was never demonstrated and its effect on protein fate is not known. We previously showed that SPY interacts physically and functionally with TCP14 and TCP15 to promote CK responses. Here, we aimed to identify how SPY regulates TCP14/15 activities and how these TCPs promote CK responses. We show that SPY activity is required for TCP14 stability. Mutation in the putative OGT domain of SPY (spy-3) stimulated TCP14 proteolysis by the 26S proteasome, which was reversed by mutation in CULLIN1 (CUL1), suggesting a role for SKP, CUL1, F-box E3 ubiquitin ligase in TCP14 proteolysis. TCP14 proteolysis in spy-3 suppressed all TCP14 misexpression phenotypes, including the enhanced CK responses. The increased CK activity in TCP14/15-overexpressing flowers resulted from increased sensitivity to the hormone and not from higher CK levels. TCP15 overexpression enhanced the response of the CK-induced synthetic promoter pTCS to CK, suggesting that TCP14/15 affect early steps in CK signaling. We propose that posttranslational modification of TCP14/15 by SPY inhibits their proteolysis and that the accumulated proteins promote the activity of the CK phosphorelay cascade in developing Arabidopsis leaves and flowers.

Cytokinins (CK) play an important role in the formation of nitrogen-fixing root nodules. It has been known for years that rhizobia secrete CK in the extracellular medium but whether they play a role in nodule formation is not known. We have examined this question using the photosynthetic Bradyrhizobium sp. strain ORS285 which is able to nodulate Aeschynomene afraspera and A. indica using a Nod-dependent or Nod-independent symbiotic process, respectively. CK profiling showed that the most abundant CK secreted by Bradyrhizobium sp. strain ORS285 are the 2MeS (2-methylthiol) derivatives of trans-zeatin and isopentenyladenine. In their pure form, these CK can activate legume CK receptors in vitro, and their exogenous addition induced nodule-like structures on host plants. Deletion of the miaA gene showed that transfer RNA degradation is the source of CK production in Bradyrhizobium sp. strain ORS285. In nodulation studies performed with A. indica and A. afraspera, the miaA mutant had a 1-day delay in nodulation and nitrogen fixation. Moreover, A. indica plants formed considerably smaller but more abundant nodules when inoculated with the miaA mutant. These data show that CK produced by Bradyrhizobium sp. strain ORS285 are not the key signal triggering nodule formation during the Nod-independent symbiosis but they contribute positively to nodule development in Aeschynomene plants.

Rhodococcus is an important industrial microorganism that possesses diverse metabolic capabilities; it also has a cell envelope, composed of an outer layer of mycolic acids and glycolipids. Selected Rhodococcus species when induced are capable of transforming nitriles to the corresponding amide by the enzyme nitrile hydratase (NHase), and subsequently to the corresponding acid via an amidase. This nitrile biochemistry has generated interest in using the rhodococci as biocatalysts. It was hypothesized that altering sugars in the growth medium might impact cell envelope components and have effects on NHase. When the primary carbon source in growth media was changed from glucose to fructose, maltose, or maltodextrin, the NHase activity increased. Cells grown in the presence of maltose and maltodextrin showed the highest activities against propionitrile, 197 and 202 units/mg cdw, respectively. Stability of NHase was also affected as cells grown in the presence of maltose and maltodextrin retained more NHase activity at 55 °C (45 and 23 %, respectively) than cells grown in the presence of glucose or fructose (19 and 10 %, respectively). Supplementation of trehalose in the growth media resulted in increased NHase stability at 55 °C, as cells grown in the presence of glucose retained 40 % NHase activity as opposed to 19 % without the presence of trehalose. Changes in cell envelope components, such mycolic acids and glycolipids, were evaluated by high-performance liquid chromatography (HPLC) and thin-layer chromatography (TLC), respectively. Changing sugars and the addition of inducing components for NHase, such as cobalt and urea in growth media, resulted in changes in mycolic acid profiles. Mycolic acid content increased 5 times when cobalt and urea were added to media with glucose. Glycolipids levels were also affected by the changes in sugars and addition of inducing components. This research demonstrates that carbohydrate selection impacts NHase activity and

Full Text Available In vitro organogenesis, one of the most common pathways leading to in vitro plant regeneration, is widely used in biotechnology and the fundamental study of plant biology. Although previous studies have constructed a complex regulatory network model for Arabidopsis in vitro organogenesis, no related study has been reported in ramie. To generate more complete observations of transcriptome content and dynamics during ramie in vitro organogenesis, we constructed a reference transcriptome library and ten digital gene expression (DGE libraries for illumina sequencing. Approximately 111.34 million clean reads were obtained for transcriptome and the DGE libraries generated between 13.5 and 18.8 million clean reads. De novo assembly produced 43,222 unigenes and a total of 5,760 differentially expressed genes (DEGs were filtered. Searching against the Kyoto Encyclopedia of Genes and Genomes Pathway database, 26 auxin related and 11 cytokinin related DEGs were selected for qRT-PCR validation of two ramie cultivars, which had high (Huazhu No. 5 or extremely low (Dazhuhuangbaima shoot regeneration abilities. The results revealed differing regulation patterns of auxin and cytokinin in different genotypes. Here we report the first genome-wide gene expression profiling of in vitro organogenesis in ramie and provide an overview of transcription and phytohormone regulation during the process. Furthermore, the auxin and cytokinin related genes have distinct expression patterns in two ramie cultivars with high or extremely low shoot regeneration ability, which has given us a better understanding of the in vitro organogenesis mechanism. This result will provide a foundation for future phytohormone research and lead to improvements of the ramie regeneration system.

Interaction between brassinosteroids (BRs) (brassinolide, BL; 24-epibrassinolide, 24-epiBL; 28-homobrassinolide, 28-homoBL; castasterone, CS; 24-epicastasterone, 24-epiCS; 28-homocastasterone, 28-homoCS) and adenine- (trans-zeatin, tZ; kinetin, Kin) as well as phenylurea-type (1,3-diphenylurea, DPU) cytokinins (CKs) in the regulation of cell number, phytohormone level and the content of chlorophyll, monosaccharide and protein in unicellular green alga Chlorella vulgaris (Trebouxiophyceae) were examined. Chlorella vulgaris exhibited sensitivity to CKs in the following order of their stimulating properties: 10 nM tZ > 100 nM Kin >1 μM DPU. Exogenously applied BRs possessed the highest biological activity in algal cells at concentration of 10 nM. Among the BRs, BL was characterized by the highest activity, while 28-homoCS - by the lowest. The considerable increase in the level of all endogenous BRs by 27-46% was observed in C. vulgaris culture treated with exogenous 10 nM tZ. It can be speculated that CKs may stimulate BR activity in C. vulgaris by inducing the accumulation of endogenous BRs. CKs interacted synergistically with BRs increasing the number of cells and endogenous accumulation of proteins, chlorophylls and monosaccharides in C. vulgaris. The highest stimulation of algal growth and the contents of analyzed biochemical parameters were observed for BL applied in combination with tZ, whereas the lowest in the culture treated with both 28-homoCS and DPU. However, regardless of the applied mixture of BRs with CKs, the considerable increase in cell number and the metabolite accumulation was found above the level obtained in cultures treated with any single phytohormone in unicellular green alga C. vulgaris.

Full Text Available Rhizome cultures of "Jiianita" Polish cultivar of Alstroemeria x hybrida were used to enhance an effectiveness of micropropagation method of new cultivars and selections. The effect of cytokinins (BAP. kinetin and 2iP, auxins (IAA, IBAand NAA, growth retardants (paclobutrazol and flurprimidol alone or in combination were studied in relation to rhizome branching. aerial shoot production and rooting of rhizome. The greatest number of aerial shoots as well as the shortest shoots were observed at the highest BAP concentration (6 mg l-1. However, the rhizonies had the poorest rooting ability. BAP at low concentrations combined with kinetin or 2iP also strongly stimulated aerial shoot formation and rhizome branching. Unfortunately. those shoots were of poor qualily. Application of BAP at low concentration with paclobutrazol (0,1-0,5 mg l-1 or flurprimidol (0,01- 1 mg l-1 in presence of 1 mg l-1 NAA resulted in high number of aerial shoots (5-6, reduction of their length and higher rooting ability of the rhizomes. Gr(wth retardants applied with NAA strongly stimulated formation of the roots but suppressed their elongation. Abbreviations: BAP - 6=benzylaminopurine; kinetin - 6-furfurylaminopurine; 2iP - 6-‌γ,γ-dime-thylallylamino]purine; IAA-indole-3-acetic acid; IBA-indole-3-butyric acid; NAA- naphthaleneacetic acid; paclobutrazol (ICI PP-333 - (2-RS,3-RS-1-(4-chlorophenyl-4-4-dimethyl-2(1,2,3-triazol-1-yl-pentan-3-ol flurprimidol (Dowelanco - α-(1-niethylethy 1-α-[4-trifluro-niethoxyphenyl]-5-pyridinemethanol.

In this study, we used a loss-of-function approach to elucidate the functions of three Arabidopsis type B response regulators (ARRs)--namely ARR1, ARR10, and ARR12--in regulating the Arabidopsis plant responses to drought. The arr1,10,12 triple mutant showed a significant increase in drought tolerance versus WT plants, as indicated by its higher relative water content and survival rate on drying soil. This enhanced drought tolerance of arr1,10,12 plants can be attributed to enhanced cell membrane integrity, increased anthocyanin biosynthesis, abscisic acid (ABA) hypersensitivity, and reduced stomatal aperture, but not to altered stomatal density. Further drought-tolerance tests of lower-order double and single mutants indicated that ARR1, ARR10, and ARR12 negatively and redundantly control plant responses to drought, with ARR1 appearing to bear the most critical function among the three proteins. In agreement with these findings, a comparative genome-wide analysis of the leaves of arr1,10,12 and WT plants under both normal and dehydration conditions suggested a cytokinin (CK) signaling-mediated network controlling plant adaptation to drought via many dehydration/drought- and/or ABA-responsive genes that can provide osmotic adjustment and protection to cellular and membrane structures. Expression of all three ARR genes was repressed by dehydration and ABA treatments, inferring that plants down-regulate these genes as an adaptive mechanism to survive drought. Collectively, our results demonstrate that repression of CK response, and thus CK signaling, is one of the strategies plants use to cope with water deficit, providing novel insight for the design of drought-tolerant plants by genetic engineering.

The plasmid profiles of virulent Rhodococcus equi strains isolated on three horse-breeding farms located in different parts of Hungary were investigated. From 49 soil samples collected on the three farms, 490 R. equi isolates (10 from each sample) were obtained and tested for the presence of 15- to 17-kDa antigens (VapA) by immunoblotting and PCR. Ninety-eight VapA-positive isolates were detected from 30 of the 49 culture-positive samples with a prevalence ranging from 13.1% to 23.2%. Of the 98 virulent isolates, 70 contained an 85-kb type I plasmid, 13 contained an 87-kb type I plasmid, and 15 contained an 85-kb type III plasmid which had been uniquely isolated from soil isolates in the United States. This study demonstrates that the virulent form of R. equi is very widespread in the soil environment of these stud farms in Hungary and the plasmid pattern is different from farm to farm.

Full Text Available The identification of pathogens of viral (Rotavirus, Coronavirus, parasitic (Toxocara spp. and bacterial (Escherichia coli, Salmonella spp., Rhodococcus equi origin shed in feces, and the virulence profile of R. equi and E. coli isolates were investigated in 200 samples of sand obtained from 40 parks, located in central region of state of Sao Paulo, Brazil, using different diagnostic methods. From 200 samples analyzed, 23 (11.5% strains of R. equi were isolated. None of the R. equi isolates showed a virulent (vapA gene or intermediately virulent (vapB gene profiles. Sixty-three (31.5% strains of E. coli were identified. The following genes encoding virulence factors were identified in E. coli: eae, bfp, saa, iucD, papGI, sfa and hly. Phylogenetic classification showed that 63 E. coli isolates belonged to groups B1 (52.4%, A (25.4% and B2 (22.2%. No E. coli serotype O157:H7 was identified. Eggs of Toxocara sp. were found in three parks and genetic material of bovine Coronavirus was identified in one sample of one park. No Salmonella spp. and Rotavirus isolates were identified in the samples of sand. The presence of R. equi, Toxocara sp, bovine Coronavirus and virulent E. coli isolates in the environment of parks indicates that the sanitary conditions of the sand should be improved in order to reduce the risks of fecal transmission of pathogens of zoonotic potential to humans in these places.

The potential application of biosurfactants to remove heavy metal ion from aqueous solution by batch technique was examined. The glycolipids type biosurfactants were grown in a media containing of 20% v/v corn oil with 7 days of fermentation by Rhodococcus rhodochrous. The biosurfactants reduced the surface tension of water of about 51% from 62 mN/m to 30 mN/m. The biosurfactant increased the E24 of water-palm oil emulsion of about 55% from 43% to 97% and could maintain this E24 value of above 50% for up to 9 days. Heavy metal ion removal, in this case cadmium ion, by crude and patially purified biosurfactants has been investigated from aqueous solution at pH 6. Adsorption capacity of Cd(II) ion by crude biosurfactant with 5 and 10 minutes of contact times were 1.74 and 1.82 mg/g, respectively. Additionally, the adsorption capacity of Cd(II) ion by partially purified biosurfactant with 5 and 10 minutes of contact times were 0.79 and 1.34 mg/g, respectively. The results demonstrated that the adsorption capacity of Cd(II) ion by crude biosurfactant was higher than that of by partially purified biosurfactant. The results suggested that the biosurfactant could be used in the removal of heavy metal ions from aqueous solution.